Anti-bmp10 antibody, and therapeutic agent for hypertension and
hypertensive diseases comprising said antibody as an active ingredient

ABSTRACT

An object of the present invention is to provide an anti-BMP10 antibody, and a therapeutic agent for hypertension and a hypertensive disease, containing the antibody as an active ingredient. The present invention relates to an anti-BMP10 monoclonal antibody or an antibody fragment thereof that binds to human BMP10 (bone morphogenetic protein 10). Further, the present invention relates to a therapeutic agent for hypertension and a hypertensive disease containing an antagonist for at least one of BMP10 and a BMP9/BMP10 heterodimer, a diagnostic agent or a pharmaceutical composition for a disease associated with human BMP10, an immunological detection method or a measurement method for human BMP10 using the antagonist, and use of the antagonist for producing a pharmaceutical composition for treating hypertension and a hypertensive disease.

TECHNICAL FIELD

The present invention relates to an anti-BMP10 monoclonal antibody or anantibody fragment thereof that binds to human BMP10 (bone morphogeneticprotein 10), a DNA encoding the antibody or the antibody fragmentthereof, a recombinant vector comprising the DNA, a transformantobtained by introducing the recombinant vector into a host cell, and amethod for producing the antibody or the antibody fragment thereof usingthe transformant.

Further, the present invention relates to a therapeutic agent forhypertension and a hypertensive disease containing an antagonist for atleast one of BMP10 and a BMP9/BMP10 heterodimer, a diagnostic agent or apharmaceutical composition for a disease associated with human BMP10, animmunological detection method or a measurement method for human BMP10using the antagonist, and use of the antagonist for producing apharmaceutical composition for treating hypertension and a hypertensivedisease.

BACKGROUND ART

BMP10 is an abbreviation of bone morphogenetic protein 10. BMP10 belongsto BMP (bone morphogenetic protein) family molecules composed of about20 types, and human BMP10 is a secretory protein composed of 424 aminoacids (Non-Patent Literatures 1 and 2). BMP10 binds to two receptors oftype I and type II so as to activate the receptors, followed byphosphorylation of Smad1/5/8, and further the Smad1/5/8 activated byphosphorylation form a complex with Smad4, and thereafter, the complextranslocates into the nucleus and functions as a transcription factor.

It is known that BMP10 is mainly expressed in a heart (Non-PatentLiteratures 2, 3, and 4), and that human BMP10 is a blood circulationfactor present in blood at a concentration of about 10 ng/mL (Non-PatentLiterature 5).

With respect to the in vivo role of BMP10, based on an analysis usingBMP10-deficient mice, it has been reported that BMP10 is an importantfactor for heart development and angiogenesis in the embryonic stage sofar (Non-Patent Literatures 6 and 7). However, the BMP10-deficient miceare embryonic lethal, and therefore, there have been no reports on theeffect of BMP10 in the adult stage. In addition, there have been noreports based on the administration of an anti-BMP10 antibody to ananimal in the adult stage.

Further, it has been reported that there is a gene mutation in BMP10 ina patient with HT-DCM that is dilated cardiomyopathy accompanied byhypertension, and the gene mutation causes hypersecretion of BMP10(Non-Patent Literature 12). In Non-Patent Literature 12, it is indicatedthat when hypertensive rats are given high salt to cause cardiachypertrophy, the expression of BMP10 mRNA is increased. However,Non-Patent Literature 12 does not describe neutralization and deletionof BMP10, and it is very difficult to predict the antihypertensiveeffect by neutralization of BMP10.

Therefore, it was extremely difficult to predict that an anti-BMP10antibody has an antihypertensive effect, a renal protective effect, anda cardioprotective effect in vivo.

As an anti-BMP10 monoclonal antibody that specifically neutralizesBMP10, MAB2926 (clone No. 462732) sold by R & D Systems, Inc., and oneantibody (clone No. 13C11) reported in the literature are known.Further, it is indicated that MAB2926 has a stronger BMP10 neutralizingactivity than 13C11 (Non-Patent Literature 8). There were no other knownspecific neutralizing antibodies against BMP10.

Hypertension is a lifestyle disease, and the number of patients withhypertension is the largest among the lifestyle diseases. There existmany drugs such as a Ca antagonist, a diuretic agent, an ACE inhibitor,and an ARB, however, it has been reported that the blood pressure couldbe appropriately controlled only in 35% of the patients in the USA(Non-Patent Literature 9). As one of the causes why hypertension cannotbe controlled, a decrease in adherence due to long administration periodis exemplified. Therefore, a novel antihypertensive drug capable ofpersistently and stably controlling the blood pressure has been awaited.

One of the forms of hypertension that is difficult to control by anexisting antihypertensive drug is salt-sensitive hypertension. Healthyindividuals excrete sodium from the kidney according to salt intake andmaintain homeostasis, but in salt-sensitive hypertensive patients, anabnormality occurs in the mechanism of sodium excretion in the kidney.In a pathological condition in which enhancement of salt sensitivityoccurs, in order to excrete the same amount of sodium as healthyindividuals, a higher blood pressure is needed, and therefore,hypertension is exhibited according to the salt intake. As a symptomatictreatment, sodium reduction therapy is carried out, however, there arenot many patients who can achieve reduction in blood pressure by sodiumreduction therapy due to a decrease in QOL by a low-sodium diet(Non-Patent Literature 10). Therefore, an antihypertensive drug with anew mechanism which targets a novel molecule and is capable of treatingsalt-sensitive hypertension has been demanded.

Poorly controlled hypertension is associated with the risk of developinga cardiovascular disease such as stroke, heart attack, heart failure, ora kidney disease. Progression of renal failure leads to blood sodiumretention, body fluid retention, activation of a blood pressureregulator, accumulation of a uremic substance, or the like, and furtherincreases blood pressure. In addition, heart failure due to body fluidretention causes an abnormality in a sympathetic nerve or a body fluidregulator as a result of a decrease in cardiac output so as to make theblood pressure control difficult. In such a manner, a vicious circle ofcardiorenal syndrome is caused in which renal failure and heart failureexacerbate hemodynamic failure such as hypertension, and hemodynamicfailure exacerbate the pathological conditions of renal failure andheart failure (Non-Patent Literature 11).

CITATION LIST Patent Literature

-   Patent Literature 1: U.S. Pat. No. 8,287,868-   Patent Literature 2: US Patent Application Publication No.    2017/0137503

Non-Patent Literature

-   Non-Patent Literature 1: FEBS Letters 586 1846-1859 (2012)-   Non-Patent Literature 2: J Biol Chem. 2011 Jul. 1; 286(26): 22785-94-   Non-Patent Literature 3: Mech Dev. 1999 February; 80(2): 181-4-   Non-Patent Literature 4: Dev Genes Evol. 2004 February; 214(2): 96-8-   Non-Patent Literature 5: Blood. 2012 Jun. 21; 119(25): 6162-6171-   Non-Patent Literature 6: Proc Natl Acad Sci USA, 2013 Jul. 16;    110(29): 11887-92-   Non-Patent Literature 7: Development. 2004 May; 131(9): 2219-2231-   Non-Patent Literature 8: Proc Natl Acad Sci USA, 2015 Jun. 23;    112(25): E3207-15-   Non-Patent Literature 9: Expert Opin Biol Ther. 2010 July; 10(7):    1077-87-   Non-Patent Literature 10: Jikken Igaku (Experimental Medicine)    33(7): 1078-1084, 2015-   Non-Patent Literature 11: Eur Rev Med Pharmacol Sci. 2014 October;    18(19): 2918-26-   Non-Patent Literature 12: Am J Physiol Heart Circ Physiol 2007    December; 293(6): H3396-403

SUMMARY OF INVENTION Technical Problem

Therefore, as a novel therapeutic agent for hypertension, a therapeuticagent capable of achieving both renal protective effect andcardioprotective effect has been awaited. In addition, as describedabove, it has been demanded that a novel therapeutic agent forhypertension has three characteristics of persistent and stable controlof blood pressure, an antihypertensive effect through a novel target,and a protective effect on kidney and heart.

An object of the present invention is to provide an anti-BMP10 antibodyand a therapeutic agent for hypertension and a hypertensive diseasecontaining the antibody as an active ingredient.

Solution to Problem

The present inventors attempted to obtain an anti-BMP10 antibody usingrats for the purpose of clarifying the in vivo effect of an anti-BMP10antibody, and successfully obtained an anti-BMP10 antibody having aremarkably improved neutralizing activity and binding activity againstBMP10 as compared with existing antibodies. In addition, they clarifiedthat while existing antibodies do not have a neutralizing activityagainst ALK1 highly expressing cells, the obtained anti-BMP10 antibodyhas a strong neutralizing activity.

Further, they found that while the existing antibodies compete withALK1, the obtained anti-BMP10 antibody has a novel mode of inhibition ofantagonizing BMPRII and endoglin. In addition, they revealed that byusing the obtained anti-BMP10 antibody, a heterodimer composed of BMP9and BMP10 is present in blood. Further, they revealed that the obtainedanti-BMP10 antibody has a strong neutralizing activity against theheterodimer composed of BMP9 and BMP10 in ALK1 highly expressing cells.

Further, by using the obtained antibody, they examined the in vivoeffect of the anti-BMP10 antibody. As a result, they found that theanti-BMP10 antibody of the present invention has a sustainedantihypertensive effect, and further remarkably improves hypertensionand a sodium excretion disorder in salt-sensitive pathologicalconditions, and has a therapeutic effect on a glomerular disorder, arenal tubular disorder, and heart diastolic dysfunction accompanyinghypertension.

Based on these findings, the present inventors considered that atherapeutic agent for hypertension and a hypertensive disease containingan anti-BMP10 antibody as an active ingredient can be provided, and thuscompleted the present invention.

That is, the present invention relates to the following (1) to (18).

(1) A monoclonal antibody comprising: a heavy chain comprisingcomplementarity determining regions (hereinafter abbreviated as CDRs) 1and 3 comprising amino acid sequences represented by SEQ ID NOs: 29 and31, respectively, and CDR2 comprising an amino acid sequence representedby SEQ ID NO: 30 or an amino acid sequence in which serine at position16 of the amino acid sequence represented by SEQ ID NO: 30 issubstituted with aspartic acid; and a light chain comprising CDRs 1 to 3comprising amino acid sequences represented by SEQ ID NOs: 32 to 34,respectively, or an antibody fragment thereof

(2) The monoclonal antibody or the antibody fragment thereof accordingto (1), comprising a light chain variable region (hereinafterabbreviated as VL) comprising any one amino acid sequence selected fromSEQ ID NOs: 71 to 87 and/or a heavy chain variable region (hereinafterabbreviated as VH) comprising any one amino acid sequence selected fromSEQ ID NOs: 70 and 88 to 98.

(3) The monoclonal antibody or the antibody fragment thereof accordingto (1) or (2) selected from the following (a) to (w):

(a) a monoclonal antibody comprising VH comprising an amino acidsequence represented by SEQ ID NO: 94 and VL comprising an amino acidsequence represented by SEQ ID NO: 73, or an antibody fragment thereof;

(b) a monoclonal antibody comprising VH comprising an amino acidsequence represented by SEQ ID NO: 95 and VL comprising an amino acidsequence represented by SEQ ID NO: 73, or an antibody fragment thereof;

(c) a monoclonal antibody comprising VH comprising an amino acidsequence represented by SEQ ID NO: 91 and VL comprising an amino acidsequence represented by SEQ ID NO: 75, or an antibody fragment thereof;

(d) a monoclonal antibody comprising VH comprising an amino acidsequence represented by SEQ ID NO: 98 and VL comprising an amino acidsequence represented by SEQ ID NO: 75, or an antibody fragment thereof;

(e) a monoclonal antibody comprising VH comprising an amino acidsequence represented by SEQ ID NO: 89 and VL comprising an amino acidsequence represented by SEQ ID NO: 77, or an antibody fragment thereof;

(f) a monoclonal antibody comprising VH comprising an amino acidsequence represented by SEQ ID NO: 97 and VL comprising an amino acidsequence represented by SEQ ID NO: 77, or an antibody fragment thereof;

(g) a monoclonal antibody comprising VH comprising an amino acidsequence represented by SEQ ID NO: 97 and VL comprising an amino acidsequence represented by SEQ ID NO: 78, or an antibody fragment thereof;

(h) a monoclonal antibody comprising VH comprising an amino acidsequence represented by SEQ ID NO: 98 and VL comprising an amino acidsequence represented by SEQ ID NO: 78, or an antibody fragment thereof;

(i) a monoclonal antibody comprising VH comprising an amino acidsequence represented by SEQ ID NO: 91 and VL comprising an amino acidsequence represented by SEQ ID NO: 79, or an antibody fragment thereof;

(j) a monoclonal antibody comprising VH comprising an amino acidsequence represented by SEQ ID NO: 95 and VL comprising an amino acidsequence represented by SEQ ID NO: 79, or an antibody fragment thereof;

(k) a monoclonal antibody comprising VH comprising an amino acidsequence represented by SEQ ID NO: 98 and VL comprising an amino acidsequence represented by SEQ ID NO: 79, or an antibody fragment thereof;

(l) a monoclonal antibody comprising VH comprising an amino acidsequence represented by SEQ ID NO: 89 and VL comprising an amino acidsequence represented by SEQ ID NO: 81, or an antibody fragment thereof;

(m) a monoclonal antibody comprising VH comprising an amino acidsequence represented by SEQ ID NO: 91 and VL comprising an amino acidsequence represented by SEQ ID NO: 81, or an antibody fragment thereof;

(n) a monoclonal antibody comprising VH comprising an amino acidsequence represented by SEQ ID NO: 95 and VL comprising an amino acidsequence represented by SEQ ID NO: 81, or an antibody fragment thereof;

(o) a monoclonal antibody comprising VH comprising an amino acidsequence represented by SEQ ID NO: 97 and VL comprising an amino acidsequence represented by SEQ ID NO: 81, or an antibody fragment thereof;

(p) a monoclonal antibody comprising VH comprising an amino acidsequence represented by SEQ ID NO: 98 and VL comprising an amino acidsequence represented by SEQ ID NO: 81, or an antibody fragment thereof;

(q) a monoclonal antibody comprising VH comprising an amino acidsequence represented by SEQ ID NO: 94 and VL comprising an amino acidsequence represented by SEQ ID NO: 85, or an antibody fragment thereof;

(r) a monoclonal antibody comprising VH comprising an amino acidsequence represented by SEQ ID NO: 95 and VL comprising an amino acidsequence represented by SEQ ID NO: 85, or an antibody fragment thereof;

(s) a monoclonal antibody comprising VH comprising an amino acidsequence represented by SEQ ID NO: 97 and VL comprising an amino acidsequence represented by SEQ ID NO: 85, or an antibody fragment thereof;

(t) a monoclonal antibody comprising VH comprising an amino acidsequence represented by SEQ ID NO: 98 and VL comprising an amino acidsequence represented by SEQ ID NO: 85, or an antibody fragment thereof;

(u) a monoclonal antibody comprising VH comprising an amino acidsequence represented by SEQ ID NO: 94 and VL comprising an amino acidsequence represented by SEQ ID NO: 87, or an antibody fragment thereof;

(v) a monoclonal antibody comprising VH comprising an amino acidsequence represented by SEQ ID NO: 97 and VL comprising an amino acidsequence represented by SEQ ID NO: 87, or an antibody fragment thereof;and

(w) a monoclonal antibody comprising VH comprising an amino acidsequence represented by SEQ ID NO: 98 and VL comprising an amino acidsequence represented by SEQ ID NO: 87, or an antibody fragment thereof.

(4) The monoclonal antibody or the antibody fragment thereof accordingto any one of (1) to (3), having a neutralizing activity against BMP10.

(5) The monoclonal antibody or the antibody fragment thereof accordingto any one of (1) to (4), having a neutralizing activity against aBMP9/BMP10 heterodimer.

(6) The monoclonal antibody or the antibody fragment thereof accordingto any one of (1) to (5), which is a genetically recombinant antibody.

(7) The antibody fragment according to any one of (1) to (6), which isan antibody fragment selected from a Fab, a Fab′, a (Fab′)₂, a singlechain antibody (scFv), a dimerized V region (diabody), a disulfidestabilized V region (dsFv), and a peptide comprising a CDR.

(8) A DNA, encoding the monoclonal antibody or the antibody fragmentthereof according to any one of (1) to (7).

(9) A recombinant vector, comprising the DNA according to (8).

(10) A transformant, obtained by introducing the recombinant vectoraccording to (9) into a host cell.

(11) A method for producing the monoclonal antibody or the antibodyfragment thereof according to any one of (1) to (7), comprising:culturing the transformant according to (10) in a culture medium, andcollecting the antibody or the antibody fragment from the culture.

(12) A therapeutic agent for hypertension and/or a hypertensive disease,comprising an antagonist for at least one of BMP10 and a BMP9/BMP10heterodimer.

(13) The therapeutic agent according to (12), which is administeredconcurrently or sequentially with a BMP9 antagonist.

(14) A therapeutic agent for hypertension and/or a hypertensive disease,the therapeutic agent comprising a BMP9 antagonist, characterized bybeing administered concurrently or sequentially with an antagonist forat least one of BMP10 and a BMP9/BMP10 heterodimer.

(15) A diagnostic agent for a disease associated with human BMP10,comprising an antagonist for at least one of BMP10 and a BMP9/BMP10heterodimer.

(16) An immunological detection method or a measurement method for humanBMP10, using an antagonist for at least one of BMP10 and a BMP9/BMP10heterodimer.

(17) A pharmaceutical composition, comprising an antagonist for at leastone of BMP10 and a BMP9/BMP10 heterodimer and a pharmacologicallyacceptable carrier.

(18) Use of an antagonist for at least one of BMP10 and a BMP9/BMP10heterodimer for producing a pharmaceutical composition for treatinghypertension and a hypertensive disease.

Advantageous Effects of Invention

The antibody of the present invention is an anti-BMP10 antibody in whichat least one of the neutralizing activity against BMP10 and the bindingactivity thereto is remarkably improved as compared with existingantibodies. Further, the antibody of the present invention has aneutralizing activity against BMP10 in ALK1 highly expressing cells.Further, the antibody of the present invention inhibits binding of humanBMP10 to human BMPRII and human endoglin.

In addition, by the antibody of the present invention, it wasdemonstrated that a heterodimer composed of human BMP9 and human BMP10is present in human blood. Further, the antibody of the presentinvention has a neutralizing activity against a heterodimer composed ofhuman BMP9 and human BMP10 in ALK1 highly expressing cells.

In addition, the antibody of the present invention has an effect ofpersistently lowering blood pressure in hypertensive pathology. Further,the antibody of the present invention remarkably improves high bloodpressure and a sodium excretion disorder in salt-sensitive hypertension.In addition, the antibody of the present invention has an improvingeffect against a renal tubulointerstitial disorder, a renal glomerulardisorder, and heart diastolic dysfunction accompanying hypertension.

The antibody of the present invention is an antibody having one or moreor all of the characteristics described above. A DNA encoding theantibody having such a characteristic, a vector comprising the DNA, atransformant obtained by introducing the vector, a method for producingthe antibody or the antibody fragment thereof using the transformant,and a therapeutic agent for hypertension and a hypertensive disease canbe provided by using the antibody or the antibody fragment thereof as anactive ingredient.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a graph comparing the BMP10 neutralizing activity of obtainedanti-BMP10 monoclonal antibodies with that of a known antibody usingId1-Luc/CHO cells. The horizontal axis represents the addedconcentration of the antibodies (μg/mL), and the vertical axisrepresents the neutralizing activity (%). Black circles indicate acontrol antibody, black squares indicate MAB2926, white circles indicate18C1 antibody, white squares indicate 12H3 antibody, and white trianglesindicate 11H10 antibody.

FIG. 2 is a figure comparing the BMP10 neutralizing activity of obtainedanti-BMP10 monoclonal antibodies with that of a known antibody usingALK1/Id1-Luc/CHO cells. The horizontal axis represents the addedconcentration of the antibodies (μg/mL), and the vertical axisrepresents the neutralizing activity (%). Black circles indicate acontrol antibody, black squares indicate MAB2926, white circles indicate18C1 antibody, white squares indicate 12H3 antibody, and white trianglesindicate 11H10 antibody.

FIG. 3 is a figure evaluating the neutralizing activity of obtained 18C1antibody against various types of BMP molecules. The vertical axisrepresents the neutralizing activity (%). White bar graphs indicate the18C1 antibody, and black bar graphs indicate a control antibody.

FIG. 4A to FIG. 4C are figures showing the effect on the systemic bloodpressure of normal rats by single administration of obtained anti-BMP10antibodies. The graph of FIG. 4A shows a change in systolic bloodpressure by single administration of 18C1 antibody. The graph of FIG. 4Bshows a change in systolic blood pressure by single administration of12H3 antibody. The graph of FIG. 4C shows a change in systolic bloodpressure by single administration of 11H10 antibody. The horizontal axisof the graph represents the elapsed time when the time at which theantibody was administered was set as 0 hour, and the vertical axisrepresents the systolic blood pressure (mmHg).

FIG. 5 is a figure showing the effect on the blood pressure ofspontaneously hypertensive rats by single administration of 18C1antibody that is an obtained anti-BMP10 antibody. The graph of FIG. 5shows a change in systolic blood pressure. The horizontal axis of thegraph represents the elapsed time when the time at which the antibodywas administered was set as 0 hour, and the vertical axis represents thesystolic blood pressure (mmHg).

FIG. 6 is a figure showing the effect of 18C1 antibody that is anobtained anti-BMP10 antibody on the systolic blood pressure of Dahlsalt-sensitive hypertensive rats. The graph of FIG. 6 shows a change insystolic blood pressure. The horizontal axis of the graph represents theweeks of age (w) of the rats, and the vertical axis represents thesystolic blood pressure (mmHg). Black circles indicate a normal dietgroup (n=6), black squares indicate a high salt group (n=12), and whitecircles indicate a high salt diet+18C1 antibody group (n=12). Error barsin the figure indicate the standard error (SE).

FIG. 7A and FIG. 7B are figures showing the effect of 18C1 antibody thatis an obtained anti-BMP10 antibody on sodium excretion in Dahlsalt-sensitive hypertensive rats. FIG. 7A shows a change in blood sodiumconcentration. The horizontal axis of the graph represents the weeks ofage (w) of the rats, and the vertical axis represents the serum sodiumconcentration (mEq). FIG. 7B shows a change in urine sodium excretionper day. The horizontal axis of the graph represents the weeks of age(w) of the rats, and the vertical axis represents the urine sodiumexcretion per day (mEq/day). Black circles indicate a normal diet group(n=6), black squares indicate a high salt group (n=12), and whitecircles indicate a high salt diet+18C1 antibody group (n=12). Error barsin the figures indicate the standard error (SE).

FIG. 8 shows a change in urine volume per day in Dahl salt-sensitivehypertensive rats by 18C1 antibody that is an obtained anti-BMP10antibody. The horizontal axis of the graph represents the weeks of age(w) of the rats, and the vertical axis represents the urine volume perbody weight per day (mL/Kg/day). Black circles indicate a normal dietgroup (n=6), black squares indicate a high salt group (n=12), and whitecircles indicate a high salt diet+18C1 antibody group (n=12). Error barsin the figure indicate the standard error (SE).

FIG. 9 is a figure showing the effect of 18C1 antibody that is anobtained anti-BMP10 antibody on a renal function in Dahl salt-sensitivehypertensive rats. FIG. 9 shows a change in urine protein excretion perday. The horizontal axis of the graph represents the weeks of age (w) ofthe rats, and the vertical axis represents the urine protein excretionper day (mg/day). Black circles indicate a normal diet group (n=6),black squares indicate a high salt group (n=12), and white circlesindicate a high salt diet+18C1 antibody group (n=12). Error bars in thefigure indicate the standard error (SE).

FIG. 10A to FIG. 10F are figures showing the effect of 18C1 antibodythat is an obtained anti-BMP10 antibody on a renal function in Dahlsalt-sensitive hypertensive rats. FIG. 10A is a figure showing renalglomerular pathology in a normal diet group in Dahl salt-sensitivehypertensive rats. FIG. 10B is a figure showing renal glomerularpathology in a high salt diet group in Dahl salt-sensitive hypertensiverats. FIG. 10C is a figure showing renal glomerular pathology in a groupin which a high salt diet was given and 18C1 antibody was administeredto Dahl salt-sensitive hypertensive rats (high salt diet+18C1). FIG. 10Dis a photograph of renal tubulointerstitial pathology in a normal dietgroup in Dahl salt-sensitive hypertensive rats. FIG. 10E is a photographof renal tubulointerstitial pathology in a high salt diet group in Dahlsalt sensitive hypertensive rats. FIG. 10F is a photograph of renaltubulointerstitial pathology in a group in which a high salt diet wasgiven and 18C1 antibody was administered to Dahl salt-sensitivehypertensive rats (high salt diet+18C1).

FIG. 11A and FIG. 11B are figures showing the effect of 18C1 antibodythat is an obtained anti-BMP10 antibody on a renal function in Dahlsalt-sensitive hypertensive rats. FIG. 11A is a graph showing theglomerular pathology scores of Dahl salt-sensitive hypertensive rats.The vertical axis represents the pathology score. FIG. 11B is a graphshowing the tubulointerstitial pathology score of Dahl salt-sensitivehypertensive rats. The vertical axis represents the pathology score.

FIG. 12A and FIG. 12B are figures showing the effect of 18C1 antibodythat is an obtained anti-BMP10 antibody on a cardiac function in Dahlsalt-sensitive hypertensive rats. FIG. 12A shows a left ventricleposterior wall thickness measured by echocardiography at 16 weeks ofage. The horizontal axis of the graph represents the type of feed andthe agent administered, and the vertical axis represents the leftventricle posterior wall thickness. FIG. 12B shows a mitral annulusvelocity e′ measured by echocardiography at 16 weeks of age. Thehorizontal axis of the graph represents the type of feed and the agentadministered, and the vertical axis represents the e′. The tested groupsare as follows: a normal diet group (n=6), a high salt group (n=12), anda high salt diet+18C1 antibody group (n=12). Error bars in the figuresindicate the standard error (SE).

FIG. 13 is a figure showing the effect of 18C1 antibody that is anobtained anti-BMP10 antibody on a cardiac function in Dahlsalt-sensitive hypertensive rats. FIG. 13 shows a value obtained bycorrecting the weight of the lung dissected and collected at 16 weeks ofage by the body weight. The vertical axis of the graph represents thelung weight to body weight ratio (lung weight (mg)/body weight (g)). Thetested groups are as follows: a normal diet group (n=6), a high saltgroup (n=12), and a high salt diet+18C1 antibody group (n=12). Errorbars in the figure indicate the standard error (SE).

FIG. 14 is a figure showing that a BMP9/BMP10 heterodimer is present inhuman blood, and 18C1 antibody that is an obtained anti-BMP10 antibodyhas a neutralizing activity against the BMP9/BMP10 heterodimer. FIG. 14shows the neutralizing activity of an anti-BMP9 antibody and ananti-BMP10 antibody on BMP in blood in ALK1/Id1-Luc/CHO cells. Thehorizontal axis represents the added concentration of the antibodies(μg/mL), and the vertical axis represents the neutralizing activity (%).White circles indicate a control antibody, black circles indicate the18C1 antibody, black squares indicate 12H3 antibody, black trianglesindicate an 11H10 antibody, black diamonds indicate 10D5 antibody thatis an anti-BMP9 antibody, white squares indicate a mixture of the 12H3antibody and the 10D5 antibody, and white triangles indicate a mixtureof the 11H10 antibody and the 10D5 antibody. The white squares and thewhite triangles are indicated by broken lines.

FIG. 15 is a figure showing that a BMP9/BMP10 heterodimer is present inblood. FIG. 15 shows the results of sandwich ELISA in which ananti-BMP10 antibody or an anti-BMP9 antibody was immobilized, humanserum was reacted therewith, and detection was carried out with anantibody obtained by biotinylating 12H3 antibody that is an anti-BMP10antibody. The vertical axis represents the absorbance (OD 450/570) inELISA, and the horizontal axis represents the final concentration (%) ofhuman serum. Black circles indicate 10D5 antibody that is an anti-BMP9antibody and was immobilized, white squares indicate 11H10 antibody thatwas immobilized, and black triangles indicate a control antibody thatwas immobilized.

FIG. 16 shows amino acid sequences of heavy chain variable regions ofhumanized antibodies designed by alteration of 18C1 antibody and aredesigned by alteration from an amino acid sequence of SEQ ID NO: 70.Regions surrounded by a frame in the sequences indicate amino acidsequences of CDRs.

FIG. 17 is a figure showing amino acid sequences of heavy chain variableregions of humanized antibodies designed by alteration of 18C1 antibodyand shows amino acid sequences designed by alteration from an amino acidsequence of SEQ ID NO: 23. Regions surrounded by a frame in thesequences indicate amino acid sequences of CDRs.

FIG. 18A and FIG. 18B are each a figure showing amino acid sequences oflight chain variable regions of humanized antibodies designed byalteration of 18C1 antibody. Regions surrounded by a frame in eachsequence indicate amino acid sequences of CDRs. FIG. 18A shows aminoacid sequences designed by alteration from an amino acid sequence of SEQID NO: 71. FIG. 18B shows amino acid sequences designed by alterationfrom an amino acid sequence of SEQ ID NO: 26.

FIG. 19 is a figure showing amino acid sequences of heavy chain variableregions of humanized antibodies designed by alteration of 18C1 antibodyand shows amino acid sequences designed by alteration from an amino acidsequence of SEQ ID NO: 70. Regions surrounded by a frame in thesequences indicate amino acid sequences of CDRs.

FIG. 20 is a graph comparing the BMP10 neutralizing activity ofanti-BMP10 humanized antibodies with that of 18C1 chimeric antibodyusing ALK1/Id1-Luc/CHO cells. The horizontal axis represents the addedconcentration of the antibodies (ng/mL), and the vertical axisrepresents the neutralizing activity (%). White squares indicateVLres02HVmut07, white triangles indicate VLres02HVmut08, black trianglesindicate a VLres04HVmut04 antibody, black circles indicate aVLres04VHres16 antibody, and white circles indicate ch18C1 antibody. Theblack circles and the black triangles are indicated by broken lines.

FIG. 21 is a graph comparing the BMP10 neutralizing activity ofanti-BMP10 humanized antibodies with that of 18C1 chimeric antibodyusing ALK1/Id1-Luc/CHO cells. The horizontal axis represents the addedconcentration of the antibodies (ng/mL), and the vertical axisrepresents the neutralizing activity (%). White squares indicateVLres06HVmut02, white triangles indicate VLres06HVmut10, black trianglesindicate a VLres07HVmut10 antibody, black circles indicate aVLres07VHres16 antibody, and white circles indicate ch18C1 antibody. Theblack circles and the black triangles are indicated by broken lines.

FIG. 22 is a graph comparing the BMP10 neutralizing activity ofanti-BMP10 humanized antibodies with that of 18C1 chimeric antibodyusing ALK1/Id1-Luc/CHO cells. The horizontal axis represents the addedconcentration of the antibodies (ng/mL), and the vertical axisrepresents the neutralizing activity (%). White squares indicateVLres08HVmut04, white triangles indicate VLres08HVmut08, black trianglesindicate a VLres08VHres16 antibody, black circles indicate aVLres10HVmut02 antibody, and white circles indicate ch18C1 antibody. Theblack circles and the black triangles are indicated by broken lines.

FIG. 23 is a graph comparing the BMP10 neutralizing activity ofanti-BMP10 humanized antibodies with that of 18C1 chimeric antibodyusing ALK1/Id1-Luc/CHO cells. The horizontal axis represents the addedconcentration of the antibodies (ng/mL), and the vertical axisrepresents the neutralizing activity (%). White squares indicateVLres10HVmut04, white triangles indicate VLres10HVmut08, black trianglesindicate a VLres10HVmut10 antibody, black circles indicate aVLres10VHres16 antibody, and white circles indicate ch18C1 antibody. Theblack circles and the black triangles are indicated by broken lines.

FIG. 24 is a graph comparing the BMP10 neutralizing activity ofanti-BMP10 humanized antibodies with that of 18C1 chimeric antibodyusing ALK1/Id1-Luc/CHO cells. The horizontal axis represents the addedconcentration of the antibodies (ng/mL), and the vertical axisrepresents the neutralizing activity (%). White squares indicateVLres14HVmut07, white triangles indicate VLres14HVmut08, black trianglesindicate a VLres14HVmut10 antibody, black circles indicate aVLres14VHres16 antibody, and white circles indicate ch18C1 antibody. Theblack circles and the black triangles are indicated by broken lines.

FIG. 25 is a graph comparing the BMP10 neutralizing activity ofanti-BMP10 humanized antibodies with that of 18C1 chimeric antibodyusing ALK1/Id1-Luc/CHO cells. The horizontal axis represents the addedconcentration of the antibodies (ng/mL), and the vertical axisrepresents the neutralizing activity (%). White squares indicateVLres16HVmut07, white triangles indicate VLres16HVmut10, black trianglesindicate a VLres16VHres16 antibody, and black circles indicate ch18C1antibody. The black circles and the black triangles are indicated bybroken lines.

FIG. 26 is a graph comparing the neutralizing activity of anti-BMP10humanized antibodies against a BMP9/BMP10 heterodimer in human bloodwith that of 18C1 chimeric antibody using ALK1/Id1-Luc/CHO cells. Thevertical axis represents the luciferase activity.

FIG. 27A and FIG. 27B are each a figure showing an effect on thesystemic blood pressure of normal rats by single administration of ananti-BMP9 antibody or a mixed solution of an anti-BMP9 antibody and ananti-BMP10 antibody. FIG. 27A shows a change in systolic blood pressureby single administration of 10D5 antibody. FIG. 27B shows a change insystolic blood pressure by single administration of a mixed solution of10D5 antibody and 11H10 antibody. White circles indicate a change on theday of administration of a vehicle (PBS), white triangles indicate achange on the day of administration of the antibody, black squaresindicate a change one day after administration of the antibody, andblack triangles indicate a change two days after administration of theantibody. The black squares and the black triangles are indicated bybroken lines. The horizontal axis of the graph represents the elapsedtime when the time at which the drug solution was administered was setas 0 hour, and the vertical axis represents the systolic blood pressure(mmHg).

FIG. 28 is a figure showing the effect on the systemic blood pressure ofnormal rats by single administration of a human BMP10 recombinantprotein. White circles indicate a change on the day of administration ofa vehicle (PBS), white triangles indicate a change on the day ofadministration of the human BMP10 recombinant protein, the horizontalaxis of the graph represents the elapsed time when the time at which thedrug solution was administered was set as 0 hour, and the vertical axisrepresents the systolic blood pressure (mmHg).

DESCRIPTION OF EMBODIMENTS

The present invention relates to a monoclonal antibody that binds tohuman BMP10. Further, the present invention relates to a monoclonalantibody that binds to a BMP9/BMP10 heterodimer.

In the present invention, an antibody that binds to human BMP10 whilecompeting with the monoclonal antibody of the present invention refersto an antibody that inhibits the binding of the monoclonal antibody ofthe present invention to human BMP10 in a desired binding assay system.

An antibody that binds to the same epitope as the epitope to which themonoclonal antibody of the present invention binds refers to an antibodythat recognizes and binds to the same sequence as the amino acidsequence of human BMP10 that is recognized by the monoclonal antibody ofthe present invention.

The human BMP10 is synthesized as a single-chain precursor protein(Pre-Pro protein) having an amino acid sequence represented by SEQ IDNO: 47. This single-chain Pre-Pro protein forms a dimer (a Pro dimer ora full-length protein dimer) through a disulfide bond between cysteineresidues present at position 388 after forming a full-length protein bycleaving off, from the amino acid sequence represented by SEQ ID NO: 2,a signal peptide region which is from position 1 to position 21 of it.

Thereafter, by a furin-like protease, cleavage occurs between amino acidresidues at positions 316 and 317 of the amino acid sequence representedby SEQ ID NO: 47, and it is divided into a propeptide region of anN-terminal side fragment (a peptide comprising an amino acid sequencefrom an amino acid at position 22 to an amino acid at position 316 ofthe amino acid sequence represented by SEQ ID NO: 47, also referred toas an N-terminal propeptide protein) and a C-terminal side fragment(also referred to as a mature region or a mature protein) composed of anamino acid sequence represented by SEQ ID NO: 48.

The mature region forms a dimer (hereinafter referred to as a maturedimer) through a disulfide bond between cysteine residues remaining atposition 72 of the amino acid sequence represented by SEQ ID NO: 48 evenafter cleavage of the propeptide region. Two molecules of the cleavedN-terminal side propeptide region form a complex with one molecule ofthis mature dimer through a non-covalent bond, and are secreted from acell in the form of the complex. Both the mature dimer and the complexin which the N-terminal propeptide region is bound to the mature dimerhave the function of BMP10.

Therefore, as the human BMP10 in the present invention, a polypeptidethat comprises an amino acid sequence (SEQ ID NO: 48) which is fromposition 317 to position 424 of the amino acid sequence represented bySEQ ID NO: 47 or GenBank Accession No. NP 055297, corresponding to themature region, and has the function of human BMP10 is mentioned.

Further, as the human BMP10 in the present invention, a polypeptide thatcomprises an amino acid sequence in which one or more amino acids aredeleted, substituted, or added in the amino acid sequence represented bySEQ ID NO: 48 and has the function of human BMP10 is mentioned. Inaddition, as the human BMP10 in the present invention, a polypeptidethat comprises an amino acid sequence having 60% or more, preferably 80%or more, more preferably 90% or more, and most preferably 95% or morehomology with the amino acid sequence represented by SEQ ID NO: 48, andhas the function of human BMP10 is mentioned. Further, as the humanBMP10 in the present invention, the mature dimer and the complex inwhich the N-terminal propeptide region is bound to the mature dimerdescribed above are also included.

The human BMP9 is synthesized as a single-chain precursor protein(Pre-Pro protein) having an amino acid sequence represented by SEQ IDNO: 66. This single-chain Pre-Pro protein forms a dimer (a Pro dimer)through a disulfide bond between cysteine residues present at position392 after cleaving off, from the amino acid sequence represented by SEQID NO: 66, a signal peptide region which is from position 1 to position22 of it.

Thereafter, by a furin-like protease, cleavage occurs between amino acidresidues at positions 319 and 320 of the amino acid sequence representedby SEQ ID NO: 66, and it is divided into a propeptide region of anN-terminal side fragment (a peptide comprising an amino acid sequencefrom an amino acid at position 23 to an amino acid at position 319 ofthe amino acid sequence represented by SEQ ID NO: 66) and a C-terminalside fragment (a mature region) composed of an amino acid sequencerepresented by SEQ ID NO: 65.

The mature region forms a dimer (hereinafter referred to as a maturedimer) through a disulfide bond between cysteine residues remaining atposition 73 of an amino acid sequence represented by SEQ ID NO: 65 evenafter cleavage of the propeptide region. Two molecules of the cleavedN-terminal side propeptide region form a complex with one molecule ofthis mature dimer through a non-covalent bond, and are secreted from acell in the form of the complex [J. Biol. Chem., 280, 26, 25111 (2005)].Both the mature dimer and the complex in which the N-terminal propeptideregion is bound to the mature dimer have the function of BMP9.

Therefore, as the human BMP9 in the present invention, a polypeptidethat contains an amino acid sequence (SEQ ID NO: 65) which is fromposition 320 to position 429 of the amino acid sequence represented bySEQ ID NO: 66 or GenBank Accession No. NP 057288, corresponding to themature region, and has the function of human BMP9 is mentioned.

Further, as the human BMP9 in the present invention, a polypeptide thatcomprises an amino acid sequence in which one or more amino acids aredeleted, substituted, or added in the amino acid sequence represented bySEQ ID NO: 65 and has the function of human BMP9 is mentioned.

In addition, as the human BMP9 in the present invention, a polypeptidethat comprises an amino acid sequence having 60% or more, preferably 80%or more, more preferably 90% or more, and most preferably 95% or morehomology with the amino acid sequence represented by SEQ ID NO: 65, andhas the function of human BMP9 is mentioned. Further, as the human BMP9in the present invention, the mature dimer and the complex in which theN-terminal propeptide region is bound to the mature dimer describedabove are also included.

The above-mentioned BMP forms not only a dimer of the same protein(hereinafter referred to as a homodimer), but also a dimer of differentproteins belonging to the BMP family (hereinafter referred to as aheterodimer). The heterodimer composed of BMP9 and BMP10 (also referredto as a BMP9/BMP10 heterodimer) in the present invention comprises boththe amino acid sequence corresponding to the BMP10 mature regionrepresented by SEQ ID NO: 48 and the amino acid sequence correspondingto the BMP9 mature region represented by SEQ ID NO: 65.

Further, in the BMP9/BMP10 heterodimer in the present invention, apolypeptide that comprises an amino acid sequence in which one or moreamino acids are deleted, substituted, or added in at least one of theamino acid sequence represented by SEQ ID NO: 48 and the amino acidsequence represented by SEQ ID NO: 65 and has the function of a humanBMP9/BMP10 heterodimer is also included. In addition, in the BMP9/BMP10heterodimer in the present invention, a polypeptide that comprises anamino acid sequence having 60% or more, preferably 80% or more, morepreferably 90% or more, and most preferably 95% or more homology with atleast one of the amino acid sequence represented by SEQ ID NO: 48 andthe amino acid sequence represented by SEQ ID NO: 65, and has thefunction of the human BMP9/BMP10 heterodimer is also included. Further,as the BMP9/BMP10 heterodimer in the present invention, a complex inwhich an N-terminal propeptide region is bound to the human BMP9/BMP10heterodimer is also included.

As the function of the above-mentioned BMP10, involvement of BMP10 inthe intracellular signal transduction is mentioned. In the intracellularsignal transduction, BMP10 binds to two receptors of type I and type IIbelonging to the TGFβ superfamily so as to activate the receptors,followed by Smad1/5/8 phosphorylation, and further, the Smad1/5/8activated by phosphorylation forms a complex with Smad4, and thereafter,the complex translocates into the nucleus and functions as atranscription factor.

As the function of the above-mentioned BMP9, involvement of BMP9 in theintracellular signal transduction is mentioned. In the intracellularsignal transduction, BMP9 binds to two receptors of type I and type IIbelonging to the TGFβ superfamily so as to activate the receptors,followed by Smad1/5/8 phosphorylation, and further, the Smad1/5/8activated by phosphorylation forms a complex with Smad4, and thereafter,the complex translocates into the nucleus and functions as atranscription factor.

Examples of the type I receptor include ALK1, ALK2, ALK3, and ALK6.Further, examples of the type II receptor include a BMP type II receptor(BMPRII), an activin type IIa receptor (ActRIIa), and an activin typeIIb receptor (ActRIIb). In addition, as a receptor other than type I andtype II, endoglin is exemplified.

As a method for obtaining the polypeptide having an amino acid sequencein which one or more amino acids are deleted, substituted, or added inat least one of the amino acid sequence represented by SEQ ID NO: 48 andthe amino acid sequence represented by SEQ ID NO: 65, for example, amethod for introducing a site-specific mutation into a gene encoding thepolypeptide having the amino acid sequence represented by SEQ ID NO: 48or SEQ ID NO: 65 using a site-specific mutagenesis method [MolecularCloning, A Laboratory Manual, Second Edition, Cold Spring HarborLaboratory Press (1989), Current Protocols in Molecular Biology, JohnWiley & Sons (1987-1997), Nucleic Acids Research, 10, 6487 (1982), Proc.Natl. Acad. Sci. USA, 79, 6409 (1982), Gene, 34, 315 (1985), Proc. Natl.Acad. Sci. USA, 82, 488 (1985)], or the like is exemplified.

The number of amino acids to be deleted, substituted, or added is notparticularly limited, but is preferably one to several tens, forexample, 1 to 20, and more preferably one to several, for example, 1 to5 amino acids.

As a gene encoding human BMP10, a base sequence represented by SEQ IDNO: 49 or GenBank Accession No. NM_014482 is exemplified. A gene that iscomposed of a nucleotide sequence in which one or more nucleotides aredeleted, substituted, or added in a nucleotide sequence represented bySEQ ID NO: 50 corresponding to a mature region therein, and contains aDNA encoding a polypeptide having the function of human BMP10 is alsoincluded in the gene encoding human BMP10 of the present invention.Further, a gene that is composed of a nucleotide sequence having atleast 60% or more, preferably 80% or more, and further more preferably95% or more homology with the nucleotide sequence represented by SEQ IDNO: 50, and contains a DNA encoding a polypeptide having the function ofhuman BMP10 is also included in the gene encoding human BMP10 of thepresent invention. In addition, a gene that is composed of a DNA whichhybridizes with a DNA having the nucleotide sequence represented by SEQID NO: 50 under stringent conditions, and that contains a DNA encoding apolypeptide having the function of human BMP10, and the like are alsoincluded in the gene encoding human BMP10 of the present invention.

The DNA which hybridizes under stringent conditions means, for example,a hybridizable DNA that is obtained by a colony hybridization method, aplaque hybridization method, a southern blot hybridization method, a DNAmicroarray method, or the like using a DNA having the nucleotidesequence represented by SEQ ID NO: 50 as a probe.

Specifically, a DNA that can be identified by washing a filter or amicroscope slide under the condition of 65° C. using an SSC solutionhaving a concentration of 0.1 to 2 times (a composition of the SSCsolution having a concentration of 1 time is composed of 150 mmol/Lsodium chloride and 15 mmol/L sodium citrate), after performinghybridization [Molecular Cloning, A Laboratory Manual, Second Edition,Cold Spring Harbor Laboratory Press (1989), Current Protocols inMolecular Biology, John Wiley & Sons (1987-1997), DNA Cloning 1: CoreTechniques, A Practical Approach, Second Edition, Oxford University(1995)] at 65° C. in the presence of 0.7 to 1.0 mol/L sodium chlorideusing a filter or a microscope slide on which a DNA derived from ahybridized colony or plaque, or a PCR product or an oligo DNA having thesequence is immobilized can be exemplified.

As the hybridizable DNA, a DNA having at least 60% or more homology,more preferably a DNA having 80% or more homology, and further morepreferably a DNA having 95% or more homology with the nucleotidesequence represented by SEQ ID NO: 50 can be exemplified.

A gene polymorphism is often recognized in a nucleotide sequence of agene encoding a protein of a eukaryote. A gene in which a small-scalemutation has occurred in a nucleotide sequence due to such apolymorphism in a gene used in the present invention is also included inthe gene encoding the human BMP10 of the present invention.

The value of homology in the present invention may be a value calculatedusing a homology search program known to those skilled in the art unlessotherwise particularly specified, however, with respect to a nucleotidesequence, a value calculated using a default parameter in BLAST [J. Mol.Biol., 215, 403 (1990)], and the like are exemplified, and with respectto an amino acid sequence, a value calculated using a default parameterin BLAST2 [Nucleic Acids Res., 25, 3389 (1997), Genome Res., 7, 649(1997),http://www.ncbi.nlm.nih.gov/Education/BLASTinfo/information3.html], andthe like are exemplified.

As for the default parameters, G (Cost to open gap) is 5 in the case ofa nucleotide sequence and 11 in the case of an amino acid sequence, −E(Cost to extend gap) is 2 in the case of a nucleotide sequence and 1 inthe case of an amino acid sequence, −q (Penalty for nucleotide mismatch)is −3, −r (reward for nucleotide match) is 1, −e (expect value) is 10,−W (wordsize) is 11 residues in the case of a nucleotide sequence and 3residues in the case of an amino acid sequence, −y [Dropoff (X) forblast extensions in bits] is 20 in the case of blastn and 7 in the caseof programs other than blastn, −X (X dropoff value for gapped alignmentin bits) is 15, and Z (final X dropoff value for gapped alignment inbits) is 50 in the case of blastn and 25 in the case of programs otherthan blastn (http://www.ncbi.nlm.nih.gov/blast/html/blastcgihelp.html).

A polypeptide composed of a partial sequence of the amino acid sequencerepresented by SEQ ID NO: 47 or GenBank accession No. NP 055297 can beproduced by a method known to those skilled in the art, and can beproduced by, for example, deleting part of the DNA encoding the aminoacid sequence represented by SEQ ID NO: 47 and culturing a transformanttransfected with an expression vector comprising the resulting DNA.

In addition, a polypeptide having an amino acid sequence in which one ormore amino acids are deleted, substituted, or added in the partialsequence of the amino acid sequence represented by SEQ ID NO: 47 orGenBank accession No. NP_055297 can be obtained by the same method asdescribed above based on the polypeptide or the DNA produced by theabove-mentioned method.

Further, a polypeptide composed of the partial sequence of the aminoacid sequence represented by SEQ ID NO: 47 or GenBank accession No.NP_055297, or a polypeptide having an amino acid sequence in which oneor more amino acids are deleted, substituted, or added in the partialsequence of the amino acid sequence represented by SEQ ID NO: 47 orGenBank accession No. NP_055297 can also be produced by a chemicalsynthesis method such as a fluorenylmethyloxycarbonyl (Fmoc) method or at-butyloxycarbonyl (tBoc) method.

With respect also to the BMP9, a polypeptide having an amino acidsequence in which one or more amino acids are deleted, substituted, oradded can be produced by the above-mentioned method or a known methodsuch as a method described in JP-A-2017-25011.

The antagonist of the present invention refers to a substance thatinhibits the activation of a receptor by inhibiting the binding of aligand to its receptor protein.

The BMP10 antagonist of the present invention refers to a substance thatinhibits the activation of its receptor by inhibiting the binding of atleast one of BMP10 and a BMP9/BMP10 heterodimer to their receptorprotein. Further, the BMP10 antagonist of the present invention refersto a substance that has a neutralizing activity against at least one ofBMP10 and a BMP9/BMP10 heterodimer. The BMP10 antagonist of the presentinvention includes the anti-BMP10 monoclonal antibody or the antibodyfragment thereof of the present invention.

The BMP9 antagonist of the present invention refers to a substance thatinhibits the activation of its receptor by inhibiting the binding ofBMP9 to its receptor protein. Further, the BMP9 antagonist of thepresent invention refers to a substance that has a neutralizing activityagainst BMP9. As the BMP9 antagonist of the invention, for example, ananti-BMP9 antibody is exemplified.

One aspect of the anti-BMP10 monoclonal antibody of the presentinvention (hereinafter also referred to as the antibody of the presentinvention or the monoclonal antibody of the present invention) or anantibody fragment thereof is an antibody or an antibody fragment thereofthat recognizes and binds to the amino acid sequence of human BMP10 or aconformation thereof. One aspect of the antibody or the antibodyfragment thereof of the present invention is an antibody or an antibodyfragment thereof that binds to the amino acid sequence of human BMP10 ora conformation thereof, inhibits the binding of BMP10 to BMPRII,inhibits the binding of BMP10 to endoglin, and does not inhibit thebinding of BMP10 to ALK1. Further, one aspect of the antibody or theantibody fragment thereof of the present invention is an antibody or anantibody fragment thereof that recognizes and binds to the amino acidsequence of a human BMP9/BMP10 heterodimer or a conformation thereof.One aspect of the antibody or the antibody fragment thereof of thepresent invention is an antibody or an antibody fragment thereof thathas a neutralizing activity against BMP10. One aspect of the antibody orthe antibody fragment thereof of the present invention is an antibody oran antibody fragment thereof that has a neutralizing activity against aBMP9/BMP10 heterodimer.

The antibody or the antibody fragment thereof of the present inventionmay be anything as long as it has one or more of the characteristicsdescribed above, but an antibody or an antibody fragment thereof thathas all the characteristics that it binds to BMP10, inhibits the bindingof BMP10 to BMPRII and the binding of BMP10 to endoglin, does notinhibit the binding of BMP10 to ALK1, has a neutralizing activityagainst BMP10, binds to a BMP9/BMP10 heterodimer, and has a neutralizingactivity against a BMP9/BMP10 heterodimer is most preferred.

Further, as one aspect of the monoclonal antibody or the antibodyfragment thereof of the present invention, an antibody or an antibodyfragment thereof that binds to human BMP10 while competing with themonoclonal antibody of the present invention is also included.Preferably, an antibody or an antibody fragment thereof that binds tohuman BMP10 while competing with the monoclonal antibody or the antibodyfragment thereof of the present invention, and has a neutralizingactivity against at least one of BMP10 and a BMP9/BMP10 heterodimer isalso included.

Further, as one aspect of the monoclonal antibody or the antibodyfragment thereof of the present invention, an antibody or an antibodyfragment thereof that binds to the same epitope as the epitope to whichthe monoclonal antibody or the antibody fragment thereof of the presentinvention binds is also included. Preferably, an antibody or an antibodyfragment thereof that binds to the same epitope as the epitope to whichthe monoclonal antibody or the antibody fragment thereof of the presentinvention binds, and has a neutralizing activity against at least one ofBMP10 and a BMP9/BMP10 heterodimer is also included.

Examples of the amino acid sequence of the human BMP10 in the presentinvention include an amino acid sequence that contains two amino acidsequences of a human BMP10 mature region represented by SEQ ID NO: 48,and forms a disulfide bond between cysteine residues at position 72.

The conformation of the human BMP10 in the present invention may be anyconformation as long as it has an equivalent conformation to aconformation that the human BMP10 comprising the amino acid sequencerepresented by SEQ ID NO: 47, GenBank accession No. NP_055297, or SEQ IDNO: 48 can take in a natural state. The conformation that the humanBMP10 can take in a natural state refers to a natural conformation ofthe human BMP10.

Examples of the amino acid sequence of the human BMP9 in the presentinvention include an amino acid sequence that contains two amino acidsequences of a human BMP9 mature region represented by SEQ ID NO: 65,and forms a disulfide bond between cysteine residues at position 73.

The conformation of the human BMP9 in the present invention may be anyconformation as long as it has an equivalent conformation to aconformation that the human BMP9 comprising the amino acid sequencerepresented by SEQ ID NO: 66, GenBank accession No. NP_057288, or SEQ IDNO: 65 can take in a natural state. The conformation that the human BMP9can take in a natural state refers to a natural conformation of thehuman BMP9.

Examples of the amino acid sequence of the human BMP9/BMP10 heterodimerin the present invention include an amino acid sequence that containsone amino acid sequence of a human BMP10 mature region represented bySEQ ID NO: 48 and one amino acid sequence of a human BMP9 mature regionrepresented by SEQ ID NO: 65, and forms a disulfide bond betweencysteine residues.

The conformation of the human BMP9/BMP10 heterodimer in the presentinvention may be any conformation as long as it has an equivalentconformation to a conformation that the human BMP9/BMP10 heterodimercomprising two amino acid sequences: the amino acid sequence representedby SEQ ID NO: 47, GenBank accession No. NP_055297, or SEQ ID NO: 48; andthe amino acid sequence represented by SEQ ID NO: 66, GenBank accessionNo. NP_057288, or SEQ ID NO: 65 can take in a natural state. Theconformation that the human BMP9/BMP10 heterodimer can take in a naturalstate refers to a natural conformation of the human BMP9/BMP10heterodimer.

As the BMPRII in the present invention, a polypeptide comprising anamino acid sequence from position 27 to position 150 corresponding to anextracellular domain of an amino acid sequence represented by SEQ ID NO:51 or GenBank Accession No. NP_001195 is exemplified.

As the ALK1 in the present invention, a polypeptide comprising an aminoacid sequence from position 22 to position 118 corresponding to anextracellular domain of an amino acid sequence represented by SEQ ID NO:52 or GenBank Accession No. NP_000011 is exemplified.

As the endoglin in the present invention, a polypeptide comprising anamino acid sequence from position 27 to position 586 corresponding to anextracellular domain of an amino acid sequence represented by SEQ ID NO:69 or GenBank Accession No. NP_001108225 is exemplified.

The ALK1 highly expressing cell in the present invention refers to acell that expresses ALK1 more than usual. The ALK1 highly expressingcell also includes a human ALK1 expressing reporter cell and anALK1/Id1-Luc/CHO cell described in the below-mentioned Example 3, 3-1).

As the antibody in the present invention, specifically, a monoclonalantibody and an antibody fragment thereof specified in the following (A)or (B) are exemplified.

(A) A monoclonal antibody that includes: a heavy chain comprisingcomplementarity determining regions (hereinafter abbreviated as CDRs) 1to 3 comprising amino acid sequences represented by SEQ ID NOS: 29 to31, respectively; and a light chain comprising CDRs 1 to 3 comprisingamino acid sequences represented by SEQ ID NOS: 32 to 34, respectively,and an antibody fragment thereof.

(B) A monoclonal antibody that includes: a heavy chain comprisingcomplementarity determining regions (hereinafter abbreviated as CDRs) 1and 3 comprising amino acid sequences represented by SEQ ID NOS: 29 and31, respectively, and CDR2 comprising an amino acid sequence (SEQ ID NO:99) in which serine at position 16 of the amino acid sequencerepresented by SEQ ID NO: 30 is substituted with aspartic acid; and alight chain comprising CDRs 1 to 3 comprising amino acid sequencesrepresented by SEQ ID NOS: 32 to 34, respectively, and an antibodyfragment thereof.

Further, as the monoclonal antibody or the antibody fragment thereof ofthe present invention, an antibody or an antibody fragment thereof thatbinds to human BMP10 while competing with the monoclonal antibody or theantibody fragment thereof can be exemplified.

Further, as the monoclonal antibody or the antibody fragment thereof ofthe present invention, a monoclonal antibody and an antibody fragmentthereof that binds to the same epitope as the epitope present in humanBMP10 to which the monoclonal antibody or the antibody fragment thereofbinds can be exemplified.

The binding activity refers to, for example, that the antibody or theantibody fragment thereof of the present invention has an activity ofbinding to the amino acid sequence of human BMP10 or the conformationthereof.

The binding activity of the antibody of the present invention can beconfirmed by for example, a known immunological detection method forhuman BMP10 or a tissue expressing human BMP10 such as an enzyme-linkedimmunosorbent assay (ELISA) using a solid-phase antigen, a methodcapable of examining the binding affinity between a specific antigen andan antibody to the specific antigen, or the like. Other than these, amethod such as surface plasmon resonance using a Biacore system(manufactured by GE Healthcare, Inc.) or the like, and isothermaltitration calorimetry using ITC (manufactured by DKSH Holding AG).

The binding dissociation constant (Kd value) of an antibody for anantigen can be determined by any method of ELISA, surface plasmonresonance, and isothermal titration calorimetry by performing aScatchard plot or an analysis according to the package insert of eachapparatus. Specifically, the Kd value can be calculated by performing ananalysis according to a single cycle kinetics calculation method(BIAevaluation Software ver. 3, manufactured by GE Healthcare, Inc.)from a sensorgram measured using a Biacore system (manufactured by GEHealthcare, Inc.).

The neutralizing activity against BMP10 of the present invention means,for example, that an antibody or an antibody fragment thereof bound tohuman BMP10 inhibits the binding of human BMP10 to a receptor so as toinhibit the activation of the receptor. The neutralizing activity can bemeasured by, for example, a method using human BMP10 receptor expressingcells, a method capable of examining the inhibition of an antibody forbinding of human BMP10 to a receptor protein, or the like.

The neutralizing activity against a human BMP9/BMP10 heterodimer of thepresent invention means, for example, that an antibody or an antibodyfragment thereof binding to a human BMP9/BMP10 heterodimer inhibits thebinding of the human BMP9/BMP10 heterodimer to its receptor so as toinhibit the activation of the receptor.

As a method for neutralizing a human BMP9/BMP10 heterodimer, forexample, a method in which an antibody or an antibody fragment thereofthat binds to human BMP9 and an antibody or an antibody fragment thereofthat binds to human BMP10 are mixed, and the binding of a humanBMP9/BMP10 heterodimer to its receptor is inhibited so as to inhibit theactivation of the receptor is exemplified. Further, a method in which byusing an antibody that binds to human BMP10 and inhibits the binding ofa human BMP9/BMP10 heterodimer to its receptor, the activation of thereceptor is inhibited is also exemplified.

As a measurement method for the neutralizing activity, for example, itcan be confirmed by a method using human BMP9/BMP10 heterodimer receptorexpressing cells, a method capable of examining the inhibition of anantibody for the binding of the human BMP9/BMP10 heterodimer to areceptor protein, or the like.

As the measurement method for the neutralizing activity using humanBMP10 receptor expressing cells or human BMP9/BMP10 heterodimer receptorexpressing cells, for example, a method such as a reporter assay fordetecting the activation of a transcription factor using an enzyme suchas luciferase is exemplified.

As a method for examining the inhibition of an antibody for the bindingof human BMP10 to a receptor protein or a human BMP9/BMP10 heterodimerto a receptor protein, for example, a method such as surface plasmonresonance using a Biacore system (manufactured by GE Healthcare, Inc.)or the like or an enzyme-linked immunosorbent assay (ELISA) isexemplified.

The binding of the antibody or the antibody fragment thereof of thepresent invention to the amino acid sequence of human BMP10 or theconformation thereof can be confirmed by a known immunological detectionmethod for human BMP10 or a tissue expressing human BMP10 such as anenzyme-linked immunosorbent assay (ELISA) using a solid-phase antigen, amethod capable of examining the binding affinity between a specificantigen and an antibody to the specific antigen, or the like.

Further, it can also be confirmed using known immunological detectionmethods [Monoclonal Antibodies-Principles and practice, Third edition,Academic Press (1996), Antibodies-A Laboratory Manual, Cold SpringHarbor Laboratory (1988), Monoclonal Antibody Experimental Manual,Kodansha Scientific books (1987)] and the like in combination.

The tissue expressing human BMP10 may be any tissue as long as itexpresses the BMP10, and for example, blood, heart, liver, and the likeare exemplified.

As the monoclonal antibody of the present invention, an antibodyproduced by a hybridoma or a genetically recombinant antibody producedby a transformant transformed with an expression vector comprising anantibody gene can be exemplified.

The monoclonal antibody is an antibody that is secreted byantibody-producing cells of a single clone, and has characteristics thatit recognizes only one epitope (also referred to as an antigenicdeterminant), and amino acid sequences (primary structures) constitutingthe monoclonal antibodies are uniform.

As the epitope, for example, a single amino acid sequence, aconformation composed of an amino acid sequence, an amino acid sequenceto which a sugar chain is bound, a conformation composed of an aminoacid sequence to which a sugar chain is bound, and the like, each ofwhich a monoclonal antibody recognizes and binds to, are exemplified.

The monoclonal antibody of the present invention binds to the amino acidsequence of human BMP10.

The epitope to which the monoclonal antibody of the present inventionbinds is included in the amino acid sequence of human BMP10 representedby SEQ ID NO: 47, and more preferably included in the amino acidsequence represented by SEQ ID NO: 48.

In the present invention, a bispecific antibody refers to an antibodyhaving two types of antigen-binding domains with differentspecificities. Each of the antigen-binding domains of the bispecificantibody may bind to different epitopes of a single antigen or may bindto different antigens.

In a single molecule of a bispecific antibody, one or moreantigen-binding domains bind to each of different epitopes of a singleantigen or different antigens, that is, each binds in a monovalent orhigher valent manner. For example, in the present invention, when asingle molecule of a bispecific antibody has one antigen-binding domainthat binds to BMP10 and one antigen-binding domain that binds to BMP9,such a bispecific antibody binds to each of BMP10 and BMP9 in amonovalent manner.

The antibody of the present invention also includes the bispecificantibody that binds to BMP9 and BMP10.

The hybridoma can be prepared by, for example, preparing theabove-mentioned human BMP10 as an antigen, inducing antibody-producingcells having antigen specificity from an animal immunized with theantigen, and further fusing the antibody-producing cells with myelomacells. The hybridoma is cultured, or the hybridoma cells areadministered to an animal to induce an ascites tumor in the animal, andthe culture solution or the ascites is separated and purified, wherebyan anti-BMP10 monoclonal antibody can be obtained.

As the animal to be immunized with the antigen, any animal can be usedas long as it can produce a hybridoma, however, a mouse, a rat, ahamster, a domestic fowl, a rabbit, or the like is preferably used. Inaddition, an antibody produced by a hybridoma prepared by obtainingcells having an antibody-producing ability from such an animal,subjecting the cells to in vitro immunization, and then fusing the cellswith myeloma cells, and the like are also included in the antibody ofthe present invention.

The genetically recombinant antibody in the present invention includesantibodies produced by gene recombination such as a human chimericantibody, a humanized antibody, a human antibody, or an antibodyfragment are also included. In the genetically recombinant antibodies,an antibody having the characteristics of a monoclonal antibody, lowantigenicity, and an extended blood half-life is preferred as atherapeutic agent. Examples of the genetically recombinant antibodyinclude antibodies obtained by altering the above-mentioned monoclonalantibody of the present invention using a gene recombinant technique.

The human chimeric antibody refers to an antibody composed of a heavychain variable region (also referred to as VH) and a light chainvariable region (also referred to as VL) of an antibody of an animalother than a human, and a heavy chain constant region (also referred toas CH) and a light chain constant region (also referred to as CL) of ahuman antibody. The human chimeric antibody of the present invention canbe produced by obtaining cDNAs encoding VH and VL from theabove-mentioned hybridoma, inserting each of the cDNAs into anexpression vector for an animal cell having genes encoding CH and CL ofa human antibody, thereby constructing a human chimeric antibodyexpression vector, and then introducing the vector into an animal cellto cause expression.

The CH of the human chimeric antibody may be any as long as it belongsto a human immunoglobulin (hereinafter referred to as hIg), butpreferably those of the hIgG class are used, and further, any one of thesubclasses belonging to the hIgG class, such as hIgG1, hIgG2, hIgG3, orhIgG4, or a variant thereof can be used. As the variant, for example, aheavy chain constant region of an IgG4 mutant obtained by substituting aSer residue at position 228 according to the EU-index in the heavy chainconstant region of hIgG4 with Pro, a Leu residue at position 235 thereinwith Glu, and an Arg residue at position 409 therein with Lys(hereinafter referred to as IgG4PE R409K) can be used. Further, the CLof the human chimeric antibody may be any as long as it belongs to hIg,and those of κ class or λ class can be used.

As the human chimeric antibody of the present invention, specifically, achimeric antibody that comprises VH of an antibody comprising an aminoacid sequence represented by SEQ ID NO: 23 and comprises VL of anantibody comprising an amino acid sequence represented by SEQ ID NO: 26is exemplified. Further, a chimeric antibody that comprises VH of anantibody comprising an amino acid sequence represented by SEQ ID NO: 24and comprises VL of an antibody comprising an amino acid sequencerepresented by SEQ ID NO: 27 is exemplified. Further, a chimericantibody that comprises VH of an antibody comprising an amino acidsequence represented by SEQ ID NO: 25 and comprises VL of an antibodycomprising an amino acid sequence represented by SEQ ID NO: 28 isexemplified.

As the humanized antibody, a human CDR-grafted antibody or a humanizedantibody by a surface reconstruction method is exemplified. Further, anantibody produced by a method in combination with a method for producingsuch a humanized antibody is also included in the humanized antibody ofthe present invention. Further, an antibody that has an amino acidsequence in which one or more amino acids are deleted, substituted, oradded in an amino acid sequence of a humanized antibody designed by sucha method, and specifically recognizes human BMP10 and/or a BMP9/BMP10heterodimer is also included in the humanized antibody of the presentinvention.

The human CDR-grafted antibody refers to an antibody in which the aminoacid sequences of CDRs of VH and VL of an antibody of an animal otherthan a human are grafted into appropriate positions of VH and VL of ahuman antibody. The human CDR-grafted antibody of the present inventioncan be produced as follows. That is, cDNAs encoding V regions, in whichthe amino acid sequences of CDRs of VH and VL of a monoclonal antibodyof an animal other than a human that specifically recognizes human BMP10and/or a BMP9/BMP10 heterodimer, and binds to the amino acid sequence ofthe human BMP10 and/or the BMP9/BMP10 heterodimer or the conformationthereof are grafted into the framework regions (hereinafter referred toas FRs) of VH and VL of an arbitrary human antibody are constructed.Subsequently, each of the cDNAs is inserted into an expression vectorfor an animal cell having genes encoding CH and CL of a human antibody,thereby constructing a human CDR-grafted antibody expression vector, andthen introducing the vector into an animal cell to cause expression.

The humanized antibody by a surface reconstruction method refers to anantibody in which an amino acid residue of FR that is considered not toaffect the binding activity of an antibody of an animal other than ahuman among the amino acids of the variable region of the antibody issubstituted with an amino acid residue considered to lower theantigenicity by a surface reconstruction method (Proc. Natl. Acad. Sci.USA, 1994, 91(3): 969-73, and Protein Engineering, 1996, 10, 895-90).The humanized antibody by a surface reconstruction method of the presentinvention can be produced as follows. That is, cDNAs encoding V regions,in which an arbitrary amino acid residue of FRs of VH and VL of amonoclonal antibody of an animal other than a human that specificallyrecognizes human BMP10 and/or a BMP9/BMP10 heterodimer, and binds to theamino acid sequence of the human BMP10 and/or the BMP9/BMP10 heterodimeror the conformation thereof is substituted with another amino acidresidue are constructed. Subsequently, each of the cDNAs is insertedinto an expression vector for an animal cell having genes encoding CHand CL of a human antibody, thereby constructing an expression vectorfor a humanized antibody by a surface reconstruction method, and thenintroducing the vector into an animal cell to cause expression.

The CH of the humanized antibody may be any as long as it belongs tohIg, but preferably those of the hIgG class are used, and further, anyone of the subclasses belonging to the hIgG class, such as hIgG1, hIgG2,hIgG3, or hIgG4, or a variant thereof can be used. As the variant, forexample, CH of IgG4PE R409K can be used. Further, the CL of thehumanized antibody may be any as long as it belongs to hIg, and those ofκ class or λ class can be used.

As the humanized antibody of the present invention, specifically, ahumanized antibody that comprises: a heavy chain comprisingcomplementarity determining regions (hereinafter abbreviated as CDRs) 1and 3 comprising amino acid sequences represented by SEQ ID NOS: 29 and31, respectively, and CDR2 comprising an amino acid sequence representedby SEQ ID NO: 30 or an amino acid sequence in which serine at position16 of the amino acid sequence represented by SEQ ID NO: 30 issubstituted with aspartic acid; and a light chain comprising CDRs 1 to 3comprising amino acid sequences represented by SEQ ID NOS: 32 to 34,respectively, is exemplified.

As the humanized antibody of the present invention, specifically, ahumanized antibody that comprises at least one of the following (a) VHand (b) VL is exemplified.

(a) VH of an antibody comprising an amino acid sequence represented bySEQ ID NO: 70 or an amino acid sequence in which at least one amino acidresidue selected from Pro at position 14, Leu at position 20, Gly atposition 27, Val at position 29, Ser at position 30, Ile at position 37,Ile at position 48, Val at position 67, Val at position 71, Asn atposition 76, Phe at position 78, Leu at position 82, Val at position 85,Val at position 92, Tyr at position 94, and Thr at position 109 in theamino acid sequence represented by SEQ ID NO: 70 is substituted withanother amino acid residue

(b) VL of an antibody comprising an amino acid sequence represented bySEQ ID NO: 71 or an amino acid sequence in which at least one amino acidresidue selected from Pro at position 7, Val at position 10, Glu atposition 12, Pro at position 14, Lys at position 16, Thr at position 19,Ile at position 20, Pro at position 41, Val at position 48, Ser atposition 75, Leu at position 81, Lys at position 82, Asp at position 88,and Tyr at position 90 in the amino acid sequence represented by SEQ IDNO: 71 is substituted with another amino acid residue

The humanized antibody of the present invention also includes anantibody in which the amino acid sequences of CDRs of VH are subjectedto substitution as specified in the following (A).

(A) the amino acid sequences of CDRs in which CDR1 of VH comprises anamino acid sequence represented by SEQ ID NO: 29 or an amino acidsequence in which an alteration of substituting Val at position 4 in theamino acid sequence represented by SEQ ID NO: 29 with Ala is introduced,CDR2 of VH comprises an amino acid sequence represented by SEQ ID NO: 30or an amino acid sequence in which an alteration of substituting Ser atposition 16 in the amino acid sequence represented by SEQ ID NO: 30 withAsp is introduced, and CDR3 of VH comprises an amino acid sequencerepresented by SEQ ID NO:

Further, the VH comprised in the humanized antibody of the presentinvention is preferably the following (1) to (14).

(1) VH comprising an amino acid sequence in which Pro at position 14,Leu at position 20, Gly at position 27, Val at position 29, Ser atposition 30, Ile at position 37, Ile at position 48, Val at position 67,Val at position 71, Asn at position 76, Phe at position 78, Leu atposition 82, Val at position 85, Val at position 92, Tyr at position 94,and Thr at position 109 in the amino acid sequence represented by SEQ IDNO: 70 are each substituted with another amino acid residue

(2) VH comprising an amino acid sequence in which Leu at position 20,Gly at position 27, Val at position 29, Ser at position 30, Ile atposition 37, Ile at position 48, Val at position 67, Val at position 71,Phe at position 78, Leu at position 82, Val at position 85, Tyr atposition 94, and Thr at position 109 in the amino acid sequencerepresented by SEQ ID NO: 70 are each substituted with another aminoacid residue

(3) VH comprising an amino acid sequence in which Leu at position 20,Gly at position 27, Ser at position 30, Ile at position 48, Val atposition 67, Val at position 71, Phe at position 78, Leu at position 82,Val at position 92, and Tyr at position 94 in the amino acid sequencerepresented by SEQ ID NO: 70 are each substituted with another aminoacid residue

(4) VH comprising an amino acid sequence in which Gly at position 27,Val at position 29, Ser at position 30, Ile at position 48, Val atposition 67, Val at position 71, Val at position 92, and Tyr at position94 in the amino acid sequence represented by SEQ ID NO: 70 are eachsubstituted with another amino acid residue

(5) VH comprising an amino acid sequence in which Leu at position 20,Gly at position 27, Ile at position 48, Val at position 71, Phe atposition 78, Leu at position 82, and Val at position 92 in the aminoacid sequence represented by SEQ ID NO: 70 are each substituted withanother amino acid residue

(6) VH comprising an amino acid sequence in which Gly at position 27,Ser at position 30, Ile at position 48, Val at position 67, Val atposition 71, and Val at position 92 in the amino acid sequencerepresented by SEQ ID NO: 70 are each substituted with another aminoacid residue

(7) VH comprising an amino acid sequence in which Ile at position 48,Val at position 67, Val at position 71, Phe at position 78, and Val atposition 92 in the amino acid sequence represented by SEQ ID NO: 70 areeach substituted with another amino acid residue

(8) VH comprising an amino acid sequence in which Gly at position 27,Val at position 71, Phe at position 78, and Val at position 92 in theamino acid sequence represented by SEQ ID NO: 70 are each substitutedwith another amino acid residue

(9) VH comprising an amino acid sequence in which Gly at position 27,Ile at position 48, Val at position 67, and Val at position 92 in theamino acid sequence represented by SEQ ID NO: 70 are each substitutedwith another amino acid residue

(10) VH comprising an amino acid sequence in which Gly at position 27,Val at position 71, Val at position 92, and Tyr at position 94 in theamino acid sequence represented by SEQ ID NO: 70 are each substitutedwith another amino acid residue

(11) VH comprising an amino acid sequence in which Gly at position 27,Val at position 71, and Val at position 92 in the amino acid sequencerepresented by SEQ ID NO: 70 are each substituted with another aminoacid residue

(12) VH comprising an amino acid sequence in which Val at position 71and Val at position 92 in the amino acid sequence represented by SEQ IDNO: 70 are each substituted with another amino acid residue

(13) VH comprising the amino acid sequence represented by SEQ ID NO: 70

(14) VH comprising an amino acid sequence in which with respect to VHspecified in the above (1) to (13), the amino acid sequences of the CDRsthereof are substituted with the amino acid sequences of the CDRsspecified in the above (A)

As the amino acid sequence of the VH, for example, an amino acidsequence in which at least one alteration selected from alterations ofsubstituting Pro at position 14 with Leu, Leu at position 20 with Ile,Gly at position 27 with Phe, Val at position 29 with Leu, Ser atposition 30 with Thr, Val at position 34 with Ala, Ile at position 37with Val, Ile at position 48 with Met, Ser at position 65 with Asp, Valat position 67 with Leu, Val at position 71 with Arg, Asn at position 76with Ser, Phe at position 78 with Val, Leu at position 82 with Met, Valat position 85 with Leu, Val at position 92 with Lys, Tyr at position 94with Phe, and Thr at position 109 with Ile is introduced in the aminoacid sequence represented by SEQ ID NO: 70 is exemplified.

Further, the VL comprised in the humanized antibody of the presentinvention is preferably the following (1) to (15).

(1) VL comprising an amino acid sequence in which Pro at position 7, Valat position 10, Glu at position 12, Pro at position 14, Lys at position16, Thr at position 19, Ile at position 20, Pro at position 41, Val atposition 48, Ser at position 75, Leu at position 81, Lys at position 82,Asp at position 88, and Tyr at position 90 in the amino acid sequencerepresented by SEQ ID NO: 71 are each substituted with another aminoacid residue

(2) VL comprising an amino acid sequence in which Pro at position 7, Valat position 10, Glu at position 12, Pro at position 14, Thr at position19, Ile at position 20, Pro at position 41, Val at position 48, Ser atposition 75, and Asp at position 88 in the amino acid sequencerepresented by SEQ ID NO: 71 are each substituted with another aminoacid residue

(3) VL comprising an amino acid sequence in which Pro at position 7, Gluat position 12, Thr at position 19, Ile at position 20, Val at position48, Ser at position 75, Leu at position 81, Asp at position 88, and Tyrat position 90 in the amino acid sequence represented by SEQ ID NO: 71are each substituted with another amino acid residue

(4) VL comprising an amino acid sequence in which Pro at position 7, Gluat position 12, Pro at position 14, Lys at position 16, Thr at position19, Pro at position 41, Ser at position 75, and Asp at position 88 inthe amino acid sequence represented by SEQ ID NO: 71 are eachsubstituted with another amino acid residue

(5) VL comprising an amino acid sequence in which Glu at position 12,Ile at position 20, Pro at position 41, Val at position 48, Ser atposition 75, Asp at position 88, and Tyr at position 90 in the aminoacid sequence represented by SEQ ID NO: 71 are each substituted withanother amino acid residue

(6) VL comprising an amino acid sequence in which Glu at position 12,Lys at position 16, Thr at position 19, Pro at position 41, Lys atposition 82, and Asp at position 88 in the amino acid sequencerepresented by SEQ ID NO: 71 are each substituted with another aminoacid residue

(7) VL comprising an amino acid sequence in which Lys at position 16,Ile at position 20, Pro at position 41, Val at position 48, Asp atposition 88, and Tyr at position 90 in the amino acid sequencerepresented by SEQ ID NO: 71 are each substituted with another aminoacid residue

(8) VL comprising an amino acid sequence in which Pro at position 14,Ile at position 20, Val at position 48, Ser at position 75, Leu atposition 81, and Asp at position 88 in the amino acid sequencerepresented by SEQ ID NO: 71 are each substituted with another aminoacid residue

(9) VL comprising an amino acid sequence in which Glu at position 12,Pro at position 14, Ile at position 20, Val at position 48, Lys atposition 82, and Tyr at position 90 in the amino acid sequencerepresented by SEQ ID NO: 71 are each substituted with another aminoacid residue

(10) VL comprising an amino acid sequence in which Thr at position 19,Pro at position 41, Val at position 48, Asp at position 88, and Tyr atposition 90 in the amino acid sequence represented by SEQ ID NO: 71 areeach substituted with another amino acid residue

(11) VL comprising an amino acid sequence in which Glu at position 12,Pro at position 14, Pro at position 41, Ser at position 75, and Asp atposition 88 in the amino acid sequence represented by SEQ ID NO: 71 areeach substituted with another amino acid residue

(12) VL comprising an amino acid sequence in which Pro at position 41,Ser at position 75, Asp at position 88, and Tyr at position 90 in theamino acid sequence represented by SEQ ID NO: 71 are each substitutedwith another amino acid residue

(13) VL comprising an amino acid sequence in which Thr at position 19,Pro at position 41, Val at position 48, and Asp at position 88 in theamino acid sequence represented by SEQ ID NO: 71 are each substitutedwith another amino acid residue

(14) VL comprising an amino acid sequence in which Ile at position 20,Val at position 48, and Asp at position 88 in the amino acid sequencerepresented by SEQ ID NO: 71 are each substituted with another aminoacid residue

(15) VL comprising the amino acid sequence represented by SEQ ID NO: 71

As the amino acid sequence of the VL, an amino acid sequence in which atleast one alteration selected from alterations of substituting Pro atposition 7 with Ser, Val at position 10 with Met, Glu at position 12with Thr, Pro at position 14 with Leu, Lys at position 16 with Ser, Thrat position 19 with Lys, Ile at position 20 with Leu, Pro at position 41with Glu or Arg, Val at position 48 with Met, Ser at position 75 withPhe, Leu at position 81 with Val, Lys at position 82 with Gln, Asp atposition 88 with Ile, and Tyr at position 90 with Phe is introduced inthe amino acid sequence represented by SEQ ID NO: 71 is exemplified.

Further, as the humanized antibody of the present invention,specifically, a humanized antibody that comprises VH specified in thefollowing (a) or (c) and/or VL specified in (b) is exemplified.

(a) VH of an antibody comprising an amino acid sequence in which atleast one amino acid residue selected from Leu at position 11, Leu atposition 14, Ser at position 19, Phe at position 27, Ser at position 68,Arg at position 71, Gln at position 77, Phe at position 79, Asn atposition 83, Leu at position 85, and His at position 107 in the aminoacid sequence represented by SEQ ID NO: 23 is substituted with anotheramino acid residue

(b) VL of an antibody comprising an amino acid sequence in which atleast one amino acid residue selected from Val at position 3, Asn atposition 8, Leu at position 14, Lys at position 19, Phe at position 75,Asn at position 80, Ile at position 83, and Ile at position 88 in theamino acid sequence represented by SEQ ID NO: 26 is substituted withanother amino acid residue

(c) VH of an antibody comprising an amino acid sequence in which atleast one amino acid residue selected from Gly at position 10, Lys atposition 13, Ser at position 19, Leu at position 20, Gly at position 27,Val at position 29, Ser at position 30, Ile at position 37, Ile atposition 48, Val at position 67, Thr at position 68, Val at position 71,Gln at position 77, Phe at position 78, Ser at position 79, Leu atposition 82, Ser at position 83, Val at position 85, Thr at position 86,Ala at position 87, Ala at position 88, Val at position 92, Tyr atposition 94, and Thr at position 109 in the amino acid sequencerepresented by SEQ ID NO: 70 is substituted with another amino acidresidue

The humanized antibody also includes an antibody in which amino acids ofCDRs of VH are substituted as follows.

VH of an antibody in which CDR1 of VH comprises an amino acid sequencerepresented by SEQ ID NO: 29 or an amino acid sequence in which analteration of substituting Val at position 4 in the amino acid sequencerepresented by SEQ ID NO: 29 with Ala is introduced, CDR2 of VHcomprises an amino acid sequence represented by SEQ ID NO: 30 or anamino acid sequence in which an alteration of substituting Ser atposition 16 in the amino acid sequence represented by SEQ ID NO: 30 withAsp is introduced, and CDR3 of VH comprises an amino acid sequencerepresented by SEQ ID NO: 31

As the amino acid sequence of the VH, for example, an amino acidsequence in which at least one alteration selected from alterations ofsubstituting Leu at position 11 with Ala, Leu at position 14 with Pro,Ser at position 19 with Asp, Phe at position 27 with Ala, Val atposition 34 with Ala, Ser at position 65 with Asp, Ser at position 68with Ala, Arg at position 71 with Lys, Gln at position 77 with Glu, Pheat position 79 with Ala, Asn at position 83 with Asp, Leu at position 85with Asp, and His at position 107 with Gln is introduced in the aminoacid sequence represented by SEQ ID NO: 23 is exemplified. Further, anamino acid sequence in which at least one alteration selected fromalterations of substituting Gly at position 10 with Asp, Lys at position13 with Gln, Ser at position 19 with Asp, Leu at position 20 with Ile,Gly at position 27 with Phe, Val at position 29 with Leu, Ser atposition 30 with Thr, Ile at position 37 with Val, Ile at position 48with Met, Ser at position 65 with Asp, Val at position 67 with Leu, Thrat position 68 with Ala, Val at position 71 with Arg, Asn at position 76with Ser, Gln at position 77 with Glu, Phe at position 78 with Val, Serat position 79 with Phe, Leu at position 82 with Met, Ser at position 83with Asp, Val at position 85 with Leu, Thr at position 86 with Gln, Alaat position 87 with Thr, Ala at position 88 with Asp, Val at position 92with Lys, Tyr at position 94 with Phe, Thr at position 109 with Ile, andLeu at position 110 with Met is introduced in the amino acid sequencerepresented by SEQ ID NO: 70 is exemplified.

As the amino acid sequence of the VL, for example, an amino acidsequence in which at least one alteration selected from alterations ofsubstituting Val at position 3 with Ala, Asn at position 8 with Asp, Leuat position 14 with Ala, Lys at position 19 with Thr, Phe at position 75with Ser, Asn at position 80 with Asp, Ile at position 83 with Val, andIle at position 88 with Val is introduced in the amino acid sequencerepresented by SEQ ID NO: 26 is exemplified.

As a specific example of the humanized antibody of the presentinvention, a humanized antibody that includes at least one of VHcomprising any one of the amino acid sequences shown in FIG. 16, FIG.17, and FIG. 19, and VL comprising any one of the amino acid sequencesshown in FIG. 18A and FIG. 18B is exemplified.

Further, as a specific example of the humanized antibody of the presentinvention, a humanized antibody that comprises VL comprising any oneamino acid sequence selected from SEQ ID NOS: 71 to 87 and/or VHcomprising any one amino acid sequence selected from SEQ ID NOS: 70 and88 to 98.

Further, as specific examples of the humanized antibody of the presentinvention, the following humanized antibodies (a) to (w) areexemplified.

(a) a humanized antibody comprising VH comprising an amino acid sequencerepresented by SEQ ID NO: 94 and VL comprising an amino acid sequencerepresented by SEQ ID NO: 73

(b) a humanized antibody comprising VH comprising an amino acid sequencerepresented by SEQ ID NO: 95 and VL comprising an amino acid sequencerepresented by SEQ ID NO: 73

(c) a humanized antibody comprising VH comprising an amino acid sequencerepresented by SEQ ID NO: 91 and VL comprising an amino acid sequencerepresented by SEQ ID NO: 75

(d) a humanized antibody comprising VH comprising an amino acid sequencerepresented by SEQ ID NO: 98 and VL comprising an amino acid sequencerepresented by SEQ ID NO: 75

(e) a humanized antibody comprising VH comprising an amino acid sequencerepresented by SEQ ID NO: 89 and VL comprising an amino acid sequencerepresented by

SEQ ID NO: 77

(f) a humanized antibody comprising VH comprising an amino acid sequencerepresented by SEQ ID NO: 97 and VL comprising an amino acid sequencerepresented by SEQ ID NO: 77

(g) a humanized antibody comprising VH comprising an amino acid sequencerepresented by SEQ ID NO: 97 and VL comprising an amino acid sequencerepresented by SEQ ID NO: 78

(h) a humanized antibody comprising VH comprising an amino acid sequencerepresented by SEQ ID NO: 98 and VL comprising an amino acid sequencerepresented by SEQ ID NO: 78

(i) a humanized antibody comprising VH comprising an amino acid sequencerepresented by SEQ ID NO: 91 and VL comprising an amino acid sequencerepresented by SEQ ID NO: 79

(j) a humanized antibody comprising VH comprising an amino acid sequencerepresented by SEQ ID NO: 95 and VL comprising an amino acid sequencerepresented by SEQ ID NO: 79

(k) a humanized antibody comprising VH comprising an amino acid sequencerepresented by SEQ ID NO: 98 and VL comprising an amino acid sequencerepresented by SEQ ID NO: 79

(l) a humanized antibody comprising VH comprising an amino acid sequencerepresented by SEQ ID NO: 89 and VL comprising an amino acid sequencerepresented by SEQ ID NO: 81

(m) a humanized antibody comprising VH comprising an amino acid sequencerepresented by SEQ ID NO: 91 and VL comprising an amino acid sequencerepresented by SEQ ID NO: 81

(n) a humanized antibody comprising VH comprising an amino acid sequencerepresented by SEQ ID NO: 95 and VL comprising an amino acid sequencerepresented by SEQ ID NO: 81

(o) a humanized antibody comprising VH comprising an amino acid sequencerepresented by SEQ ID NO: 97 and VL comprising an amino acid sequencerepresented by SEQ ID NO: 81

(p) a humanized antibody comprising VH comprising an amino acid sequencerepresented by SEQ ID NO: 98 and VL comprising an amino acid sequencerepresented by SEQ ID NO: 81

(q) a humanized antibody comprising VH comprising an amino acid sequencerepresented by SEQ ID NO: 94 and VL comprising an amino acid sequencerepresented by SEQ ID NO: 85

(r) a humanized antibody comprising VH comprising an amino acid sequencerepresented by SEQ ID NO: 95 and VL comprising an amino acid sequencerepresented by SEQ ID NO: 85

(s) a humanized antibody comprising VH comprising an amino acid sequencerepresented by SEQ ID NO: 97 and VL comprising an amino acid sequencerepresented by SEQ ID NO: 85

(t) a humanized antibody comprising VH comprising an amino acid sequencerepresented by SEQ ID NO: 98 and VL comprising an amino acid sequencerepresented by SEQ ID NO: 85

(u) a humanized antibody comprising VH comprising an amino acid sequencerepresented by SEQ ID NO: 94 and VL comprising an amino acid sequencerepresented by SEQ ID NO: 87

(v) a humanized antibody comprising VH comprising an amino acid sequencerepresented by SEQ ID NO: 97 and VL comprising an amino acid sequencerepresented by SEQ ID NO: 87

(w) a humanized antibody comprising VH comprising an amino acid sequencerepresented by SEQ ID NO: 98 and VL comprising an amino acid sequencerepresented by SEQ ID NO: 87

The human antibody essentially refers to an antibody that is naturallypresent in the human body, but also includes antibodies that areobtained from a human antibody phage library and a humanantibody-producing transgenic animal, each of which is produced due tothe recent advancement of genetic engineering, cellular engineering, ordevelopmental engineering technology, and the like.

As for the antibody that is naturally present in the human body, forexample, human peripheral blood lymphocytes are isolated and infectedwith an EB virus or the like so as to immortalize the lymphocytes,followed by cloning, whereby a lymphocyte that produces the antibody canbe cultured, and then the antibody can be purified from the culturesupernatant.

The human antibody phage library is a library in which an antibodyfragment such as a Fab or an scFv is expressed on the surfaces of phagesby inserting an antibody gene prepared from a human B cell into a phagegene. It is possible to collect a phage that expresses an antibodyfragment having a desired antigen-binding activity on the surfacethereof from the library using a binding activity to a substrate ontowhich an antigen is immobilized as an index. The antibody fragment canfurther also be converted into a human antibody molecule composed of twocomplete H chains and two complete L chains using a genetic engineeringtechnique.

The human antibody-producing transgenic animal means an animal in whicha human antibody gene is incorporated into a cell. Specifically, forexample, a human antibody-producing transgenic mouse can be produced byintroducing a human antibody gene into a mouse ES cell, implanting theES cell to an early embryo of a mouse and then allowing the embryo todevelop. A human antibody from a human antibody-producing transgenicanimal can be produced by obtaining a human antibody-producing hybridomausing a usual hybridoma production method that is performed for ananimal other than a human, and culturing the hybridoma, therebyproducing and accumulating the human antibody in the culturesupernatant.

A monoclonal antibody or an antibody fragment thereof, in which one ormore amino acids are deleted, added, substituted, or inserted in theamino acid sequence constituting the antibody or the antibody fragmentdescribed above, and which has the same activity as the antibody or theantibody fragment thereof described above is also included in themonoclonal antibody or the antibody fragment thereof of the presentinvention.

The number of amino acids to be deleted, substituted, inserted, and/oradded is one or more, and is not particularly limited, and is a numbersuch that deletion, substitution, insertion or addition can be carriedout using a well-known technique such as a site-specific mutagenesismethod [Molecular Cloning, 2nd Edition, Cold Spring Harbor LaboratoryPress (1989), Current Protocols in Molecular Biology, John Willy & Sons(1987-1997), Nucleic Acids Research, 10, 6487 (1982), Proc. Natl. Acad.Sci., USA, 79, 6409 (1982), Gene, 34, 315 (1985), Nucleic AcidsResearch, 13, 4431 (1985), Proc. Natl. Acad. Sci. USA, 82, 488 (1985)].For example, it is preferably one to several tens, more preferably 1 to20, further more preferably 1 to 10, and particularly preferably 1 to 5.

The above description that one or more amino acid residues in the aminoacid sequence of the antibody are deleted, substituted, inserted, oradded indicates as follows. That is, the description means that there isa deletion, substitution, insertion, or addition of one or a pluralityof amino acid residues in arbitrary one amino acid sequence or aplurality of amino acid sequences in the same sequence. Further, such adeletion, substitution, insertion, or addition may sometimes occursimultaneously, and the amino acid residues to be substituted, inserted,or added may be either a natural type or an unnatural type.

Examples of the natural amino acid residue include L-alanine,L-asparagine, L-aspartic acid, L-glutamine, L-glutamic acid, glycine,L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine,L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan,L-tyrosine, L-valine, L-cysteine, and the like.

Hereinafter, preferred examples of mutually substitutable amino acidresidues are shown. Amino acid residues included in the same group canbe mutually substituted.

group A: leucine, isoleucine, norleucine, valine, norvaline, alanine,2-aminobutanoic acid, methionine, O-methylserine, t-butyl glycine,t-butyl alanine, and cyclohexylalanine

group B: aspartic acid, glutamic acid, isoaspartic acid, isoglutamicacid, 2-aminoadipic acid, and 2-aminosuberic acid

group C: asparagine and glutamine

group D: lysine, arginine, ornithine, 2,4-diaminobutanoic acid, and2,3-diaminopropionic acid

group E: proline, 3-hydroxyproline, and 4-hydroxyproline

group F: serine, threonine, and homoserine

group G: phenylalanine and tyrosine

In the present invention, examples of the antibody fragment include aFab, a F(ab′)₂, a Fab′, a single chain antibody (scFv), a dimerized Vregion (diabody), a disulfide stabilized V region (dsFv), a peptidecomprising a CDR and the like.

The Fab is an antibody fragment, which has a molecular weight of about50,000 and has an antigen-binding activity, and in which about a half ofan H chain at the N-terminal side of the fragments obtained by treatingIgG with papain that is a protease (cleaved at an amino acid residue atposition 224 in the H chain) and the entire L chain are bound through adisulfide bond.

The Fab of the present invention can be obtained by treating themonoclonal antibody of the present invention with papain. Further, theFab can also be produced by inserting a DNA encoding a Fab of theantibody into an expression vector for a prokaryote or an expressionvector for a eukaryote, and then introducing the vector into aprokaryote or a eukaryote to cause expression.

The F(ab′)₂ is a fragment, which is formed by binding two Fab regionsobtained by degrading a lower part of a disulfide bond in a hinge regionof IgG with pepsin that is a protease through a hinge portion, and has amolecular weight of about 100,000, and has an antigen-binding activity.

The F(ab′)₂ of the present invention can be obtained by treating themonoclonal antibody of the present invention with pepsin. Further, theF(ab′)₂ can also be produced by binding the following Fab′ through athioether bond or a disulfide bond.

The Fab′ is an antibody fragment, which is obtained by cleaving adisulfide bond in a hinge region of the above-mentioned F(ab′)₂, and hasa molecular weight of about 50,000 and has an antigen-binding activity.The Fab′ of the present invention can be obtained by treating theF(ab′)₂ of the present invention with a reducing agent such asdithiothreitol. Further, the Fab′ can also be produced by inserting aDNA encoding a Fab′ fragment of the antibody into an expression vectorfor a prokaryote or an expression vector for a eukaryote, and thenintroducing the vector into a prokaryote or a eukaryote to causeexpression.

The scFv is a VH-P-VL or VL-P-VH polypeptide in which one VH and one VLare linked using an appropriate peptide linker (hereinafter referred toas P), and is an antibody fragment having an antigen-binding activity.

The scFv of the present invention can be produced by obtaining cDNAsencoding VH and VL of the monoclonal antibody of the present invention,constructing a DNA encoding the scFv, inserting the DNA into anexpression vector for a prokaryote or an expression vector for aeukaryote, and then introducing the expression vector into a prokaryoteor a eukaryote to cause expression.

The diabody is an antibody fragment in which an scFv is dimerized, andis an antibody fragment having a divalent antigen-binding activity. Thedivalent antigen-binding activity can be the same or one can be adifferent antigen binding activity.

The diabody of the present invention can be produced by obtaining cDNAsencoding VH and VL of the monoclonal antibody of the present invention,constructing a DNA encoding an scFv so that the length of the amino acidsequence of a peptide linker is 8 residues or less, inserting the DNAinto an expression vector for a prokaryote or an expression vector for aeukaryote, and then introducing the expression vector into a prokaryoteor a eukaryote to cause expression.

The dsFv refers to a fragment obtained by binding polypeptides, in whichone amino acid residue in each of VH and VL is substituted with acysteine residue, through a disulfide bond between the cysteineresidues. The amino acid residue to be substituted with a cysteineresidue can be selected based on prediction of the conformation of theantibody according to a known method [Protein Engineering, 7, 697(1994)].

The dsFv of the present invention can be produced by obtaining cDNAsencoding VH and VL of the monoclonal antibody of the present invention,constructing a DNA encoding a dsFv, inserting the DNA into an expressionvector for a prokaryote or an expression vector for a eukaryote, andthen introducing the expression vector into a prokaryote or a eukaryoteto cause expression.

The peptide comprising a CDR is configured to include at least one ormore regions of CDRs of VH or VL. In a peptide comprising a plurality ofCDRs, the CDRs can be bound directly or through an appropriate peptidelinker.

The peptide comprising a CDR of the present invention can be produced byconstructing DNAs encoding CDRs of VH and VL of the monoclonal antibodyof the present invention, inserting the DNAs into an expression vectorfor a prokaryote or an expression vector for a eukaryote, and thenintroducing the expression vector into a prokaryote or a eukaryote tocause expression. In addition, the peptide comprising a CDR can also beproduced by a chemical synthesis method such as an Fmoc method or a tBocmethod.

As the monoclonal antibody or the antibody fragment thereof of thepresent invention, a derivative of the antibody or the antibody fragmentthereof in which a radioisotope, a low-molecular weight agent, ahigh-molecular weight agent, a protein, or the like is bound to themonoclonal antibody or the antibody fragment thereof of the presentinvention chemically or in a genetic engineering manner is included.When the derivative of the antibody or the antibody fragment thereof isused for a detection method or a quantification method, as a reagent fordetection or a reagent for quantification, examples of the agent thatbinds to the monoclonal antibody or the antibody fragment thereof of thepresent invention include a labeling substance to be used for a usualimmunological detection or measurement method.

The derivative of the antibody or the antibody fragment thereof in thepresent invention can be produced by binding a radioisotope, alow-molecular weight agent, a high-molecular weight agent, a protein, orthe like to the N-terminal side or the C-terminal side of an H chain oran L chain of the monoclonal antibody or the antibody fragment thereofof the present invention, an appropriate substituent or side chain inthe antibody or the antibody fragment thereof, further, a sugar chain orthe like in the monoclonal antibody or the antibody fragment thereofusing a chemical method [Introduction to Antibody Engineering, ChijinShokan Co. Ltd. (1994)].

Further, the derivative of the antibody or the antibody fragment thereofin the present invention can be produced by a genetic engineeringtechnique in which a DNA encoding the monoclonal antibody or theantibody fragment thereof of the present invention is ligated to a DNAencoding a protein intended to be bound, the resultant is inserted intoan expression vector, and the expression vector is introduced into anappropriate host cell to cause expression.

Examples of the radioisotope include ¹³¹I, ¹²⁵I, ⁹⁰Y, ⁶⁴Cu, ⁹⁹Tc, ⁷⁷Lu,²¹¹At, and the like. The radioisotope can be directly bound to theantibody by a chloramine T method or the like. In addition, a substancethat chelates the radioisotope may be bound to the antibody. Examples ofthe chelating agent include1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid(MX-DTPA) and the like.

Examples of the low-molecular weight agent include a luminescentsubstance such as an acridinium ester or lophine, or a fluorescentsubstance such as fluorescein isothiocyanate (FITC) ortetramethylrhodamine isothiocyanate (RITC), and the like.

Examples of a method for binding a low-molecular weight agent to theantibody include a method for binding the agent to an amino group of theantibody through glutaraldehyde, a method for binding an amino group ofthe agent to a carboxyl group of the antibody through a water-solublecarbodiimide, and the like.

Examples of the high-molecular weight agent include polyethylene glycol(hereinafter referred to as PEG), albumin, dextran, polyoxyethylene, astyrene-maleic acid copolymer, polyvinylpyrrolidone, a pyran copolymer,hydroxypropyl methacrylamide, and the like. By binding such ahigh-molecular weight compound to the antibody or the antibody fragment,an effect such as (1) improvement of the stability against variouschemical, physical or biological factors, (2) significant extension ofthe blood half-life, or (3) elimination of immunogenicity or suppressionof antibody production is expected [Bioconjugate pharmaceutical product,Hirokawa-Shoten Ltd. (1993)]. Examples of a method for binding PEG tothe antibody include a method for reacting with a PEGylation reagent,and the like [Bioconjugate pharmaceutical product, Hirokawa-Shoten Ltd.(1993)]. Examples of the PEGylation reagent include a modifying agent toan e-amino group of lysine (JP-A-S61-178926), a modifying agent to acarboxyl group of aspartic acid and glutamic acid (JP-A-S56-23587), amodifying agent to a guanidino group of arginine (JP-A-H2-117920), andthe like.

Examples of the protein include enzymes such as alkaline phosphatase,peroxidase, or luciferase, and the like.

The present invention relates to a therapeutic agent for hypertensionand a hypertensive disease comprising a BMP10 antagonist. Further, atherapeutic agent for hypertension and a hypertensive disease whichcontains a BMP10 antagonist and is characterized by being administeredconcurrently or sequentially with a BMP9 antagonist is also included inthe present invention. In addition, a therapeutic agent for hypertensionand a hypertensive disease which contains a BMP9 antagonist and ischaracterized by being administered concurrently or sequentially with aBMP10 antagonist is also included in the present invention.

The BMP10 antagonist of the present invention includes the anti-BMP10monoclonal antibody and the antibody fragment thereof of the presentinvention as described above.

The hypertension includes all cases where the blood pressure exceeds thenormal range, but particularly, salt-sensitive hypertension and the likeare exemplified. Note that the normal range refers to a systolic bloodpressure less than 140 mmHg and a diastolic blood pressure less than 90mmHg.

The hypertensive disease includes hypertension itself and a complicationresulting from persistent high blood pressure. The hypertension can bedivided into essential hypertension whose cause cannot be specified andsecondary hypertension due to a specific cause. There is particularlysalt-sensitive hypertension as a disease considered as the cause ofessential hypertension.

Examples of the secondary hypertension include renovascularhypertension, renal parenchymal hypertension, primary aldosteronism,sleep apnea syndrome, pheochromocytoma, Cushing's syndrome, drug-inducedhypertension, pregnancy-induced hypertension, aortic coarctation,hypothyroidism, hyperthyroidism, hyperparathyroidism, brain stemvascular compression, and the like. In addition, additional examples ofthe hypertensive disease include a sodium excretion disorder, a renaltubulointerstitial disorder, a renal glomerular disorder, and heartdiastolic dysfunction which accompany hypertension, and the like.

Examples of the complication resulting from persistent high bloodpressure include cerebral infarction, cerebral hemorrhage, subarachnoidhemorrhage, arteriosclerosis, angina, myocardial infarction, congestiveheart failure (for example, systolic heart failure, diastolic heartfailure, etc.), cardiomyopathy (for example, dilated cardiomyopathy,hypertrophic cardiomyopathy, restrictive cardiomyopathy, etc.), rightheart failure, a chronic kidney disease (for example, diabeticnephropathy, nephrosclerosis, a polycystic kidney disease, chronicglomerulonephritis, tubulointerstitial nephritis, etc.), acute renaldysfunction (for example, rapidly progressive glomerulonephritis, acutetubular necrosis, etc.), aortic dissection, aortic aneurysm, retinalhemorrhage, and the like.

The therapeutic agent of the present invention contains the monoclonalantibody or the antibody fragment thereof of the present inventiondescribed above as an active ingredient.

The pharmaceutical composition of the present invention contains themonoclonal antibody or the antibody fragment thereof of the presentinvention described above as an active ingredient.

The pharmaceutical composition of the present invention contains aphysiologically acceptable diluent or carrier, and may be a mixture withanother antibody or another agent such as an antibiotic. As a suitablecarrier, for example, physiological saline, phosphate buffered saline, aphosphate buffered saline glucose solution, and buffered physiologicalsaline are exemplified, but it is not limited thereto. Alternatively,the antibody is lyophilized (freeze-dried) and may be reconstructed andused when needed by adding an aqueous buffer solution as describedabove.

Examples of the route of administration include oral administration orparenteral administration such as intraoral, intra-airway, intrarectal,subcutaneous, intramuscular, and intravenous administration, andintravenous administration is preferred. As for the dosage form, it canbe administered in any of various forms, and examples of the forminclude a spray, a capsule, a tablet, a granule, a syrup, an emulsion, asuppository, an injection, an ointment, a tape, and the like.

A liquid preparation such as an emulsion or a syrup can be producedusing, for example, water, a saccharide such as sucrose, sorbitol, orfructose, a glycol such as polyethylene glycol or propylene glycol, anoil such as sesame oil, olive oil, or soybean oil, a preservative suchas a p-hydroxybenzoic acid ester, a flavor such as strawberry flavor orpeppermint, or the like, as an additive.

A capsule, a tablet, a powder, a granule, or the like can be producedusing, for example, an excipient such as lactose, glucose, sucrose, ormannitol, a disintegrating agent such as starch or sodium alginate, alubricant such as magnesium stearate or talc, a binder such as polyvinylalcohol, hydroxypropyl cellulose, or gelatin, a surfactant such as afatty acid ester, a plasticizer such as glycerin, or the like as anadditive. In an injection, water, a saccharide such as sucrose,sorbitol, xylose, trehalose, or fructose, a sugar alcohol such asmannitol, xylitol, or sorbitol; a buffer solution such as a phosphatebuffer solution, a citrate buffer solution, or a glutamate buffersolution; a surfactant such as a fatty acid ester, or the like can beused as an additive.

Examples of the pharmaceutical preparation suitable for parenteraladministration include an injection, a suppository, a spray, and thelike. In the case of an injection, it is generally provided in a stateof a unit dose ampoule or a multiple dose container. It may be in theform of a powder to be redissolved in a suitable carrier, for example,sterile pyrogen-free water upon use. Such a dosage form usually containsan additive such as an emulsifying agent or a suspending agent that isgenerally used for formulation in such a composition.

Examples of an injection method include intravenous infusion,intravenous injection, intramuscular injection, intraperitonealinjection, subcutaneous injection, intradermal injection, and the like.In addition, the dose thereof varies depending on the age of anadministration subject, the route of administration, and administrationfrequency, and can be changed in a wide range.

A suppository is prepared using a carrier such as cacao butter, ahydrogenated fat, or carboxylic acid. Further, a spray can also beprepared using the antibody of the present invention or a functionalfragment of the antibody itself, or is prepared using a carrier whichdoes not stimulate the buccal and airway mucous membranes of a recipient(patient) and disperses the antibody or the functional fragment of theantibody as fine particles so as to facilitate absorption thereof, orthe like.

Specific examples of the carrier include lactose, glycerin, and thelike. It can also be formulated into a preparation such as an aerosol ora dry powder according to the property of the antibody or the functionalfragment of the antibody or the property of the carrier to be used.Further, a component exemplified as the additive for the oralpreparation can also be added to such a parenteral preparation.

The dose thereof varies depending on the symptoms, age, body weight, orthe like, however, in general, in the case of oral administration, itcan be administered to an adult at a daily dose of about 0.01 mg to 1000mg once or in divided doses. Further, in the case of parenteraladministration, it can be administered at a dose of about 0.01 mg to1000 mg through subcutaneous injection, intramuscular injection, orintravenous injection.

The therapeutic agent comprising the antibody or the antibody fragmentthereof of the present invention or a derivative thereof may containonly the antibody or the antibody fragment thereof, or a derivativethereof as an active ingredient, however, in general, it is preferablyprovided as a pharmaceutical preparation produced by mixing it togetherwith one or more pharmacologically acceptable carriers using anarbitrary method known in the technical field of pharmaceutics.

As the route of administration, it is preferred to use the mosteffective route in the treatment, and examples thereof include oraladministration or parenteral administration such as intraoral,intra-airway, intrarectal, subcutaneous, intramuscular, or intravenousadministration, and preferred examples thereof include intravenousadministration or subcutaneous administration.

Examples of the dosage form include a spray, a capsule, a tablet, apowder, a granule, a syrup, an emulsion, a suppository, an injection, anointment, a tape, and the like.

Further, the present invention relates to an immunological detection ormeasurement method for BMP10, including a monoclonal antibody or anantibody fragment thereof that specifically recognizes and binds to theamino acid sequence of BMP10 or the conformation thereof as an activeingredient.

Examples of a method for detecting or measuring the amount of BMP10 inthe present invention include arbitrary known methods. For example, animmunological detection or measurement method and the like areexemplified.

The immunological detection or measurement method is a method fordetecting or measuring the amount of an antibody or the amount of anantigen using a labeled antigen or antibody. Examples of theimmunological detection or measurement method include a radioimmunoassaymethod (MA), an enzyme immunoassay method (EIA or ELISA), a fluorescenceimmunoassay method (FIA), a luminescent immunoassay method, a Westernblotting method, a physicochemical method, and the like.

Hereinafter, a method for producing the antibody of the presentinvention, a therapeutic method for a disease, and a diagnostic methodfor a disease will be specifically described.

1. Method for Producing Monoclonal Antibody (1) Preparation of Antigen

BMP10 to serve as an antigen or a tissue expressing BMP10 can beobtained by introducing an expression vector containing a cDNA encodingthe full length of BMP10 or a partial length thereof into E. coli,yeast, an insect cell, an animal cell, or the like. In addition, BMP10can be obtained by purifying BMP10 from a human tissue in which BMP10 isexpressed in a large amount. In addition, the tissue or the like canalso be used as an antigen as it is. Further, a synthetic peptide havinga partial sequence of BMP10 is prepared by a chemical synthesis methodsuch as an Fmoc method or a tBoc method and can also be used as anantigen.

BMP10 used in the present invention can be produced using a methoddescribed in Molecular Cloning, A Laboratory Manual, Second Edition,Cold Spring Harbor Laboratory Press (1989), or Current Protocols InMolecular Biology, John Wiley & Sons (1987-1997), or the like, forexample, by expressing a DNA encoding the BMP10 in a host cell using thefollowing method.

First, a recombinant vector is produced by inserting a full-length cDNAcontaining a region encoding BMP10 downstream of a promoter in anappropriate expression vector. A DNA fragment that is prepared based onthe full-length cDNA and has an appropriate length containing a regionencoding a polypeptide may be used in place of the full-length cDNA.Subsequently, by introducing the obtained recombinant vector into a hostcell suitable for the expression vector, a transformant that producesthe polypeptide can be obtained.

As the expression vector, any vector can be used as long as it canautonomously replicate or can be integrated into a chromosome in a hostcell to be used, and contains an appropriate promoter at a positioncapable of transcribing a DNA encoding the polypeptide.

As the host cell, any cell can be used as long as it can express atarget gene such as a microorganism belonging to the genus Escherichiasuch as E. coli, yeast, an insect cell, or an animal cell.

When a prokaryote such as E. coli is used as the host cell, therecombinant vector is preferably a vector that can autonomouslyreplicate in the prokaryote, and also contains a promoter, a ribosomalbinding sequence, a DNA containing a region encoding BMP10, and atranscription termination sequence. In addition, the transcriptiontermination sequence is not necessarily needed for the recombinantvector, however, it is preferred that the transcription terminationsequence is located immediately downstream of a structural gene.Further, the recombinant vector may contain a gene that controls thepromoter.

As the recombinant vector, it is preferred to use a plasmid in which adistance between a Shine-Dalgarno sequence (also referred to as SDsequence), which is a ribosomal binding sequence, and a start codon isadjusted to an appropriate distance (for example, 6 to 18 bases).

In addition, in the base sequence of the DNA encoding the BMP10, it ispossible to substitute a nucleotide so that a codon becomes optimum forexpression in a host, and as a result, the production rate of the targetBMP10 can be improved.

As the expression vector, any vector can be used as long as it canexhibit its function in a host cell to be used, and examples thereofinclude pBTrp2, pBTac1, pBTac2 (all manufactured by Roche DiagnosticsK.K.), pKK233-2 (manufactured by Pharmacia Corporation), pSE280(manufactured by Invitrogen, Inc.), pGEMEX-1 (manufactured by PromegaCorporation), pQE-8 (manufactured by QIAGEN, Inc.), pKYP10(JP-A-S58-110600), pKYP200 [Agricultural Biological Chemistry, 48, 669(1984)], pLSA1 [Agric. Biol. Chem., 53, 277 (1989)], pGEL1 [Proc. Natl.Acad. Sci. USA, 82, 4306 (1985)], pBluescript II SK(−) (manufactured byStratagene Corporation), pTrs30 [prepared from E. coli JM109/pTrS30(FERM BP-5407)], pTrs32 [prepared from E. coli JM109/pTrS32 (FERMBP-5408)], pGHA2 [prepared from E. coli IGHA2 (FERM BP-400),JP-A-S60-221091], pGKA2 [prepared from E. coli IGKA2 (FERM BP-6798),JP-A-S60-221091], pTerm2 (U.S. Pat. Nos. 4,686,191, 4,939,094,5,160,735), pSupex, pUB110, pTP5, pC194, pEG400 [J. Bacteriol., 172,2392 (1990)], pGEX (manufactured by Pharmacia Corporation), pET System(manufactured by Novagen, Inc.), pME18SFL3, and the like.

As the promoter, any promoter may be used as long as it can exhibit itsfunction in a host cell to be used. Examples thereof include promotersderived from E. coli, a phage, or the like such as a trp promoter(Ptrp), a lac promoter, a PL promoter, a PR promoter, or a T7 promoter.In addition, it is also possible to use an artificially designed andaltered promoter such as a tandem promoter in which two Ptrp promotersare linked in tandem, a tac promoter, a lacT7 promoter, or a let Ipromoter.

Examples of the host cell include E. coli XL-1 Blue, E. coli XL2-Blue,E. coli DH1, E. coli MC1000, E. coli KY3276, E. coli W1485, E. coliJM109, E. coli HB101, E. coli No. 49, E. coli W3110, E. coli NY49, E.coli DH5α, and the like.

As a method for introducing a recombinant vector into a host cell, anymethod can be used as long as it is a method for introducing a DNA intoa host cell to be used, and examples thereof include a method usingcalcium ions [Proc. Natl. Acad. Sci. USA, 69, 2110 (1972), Gene, 17, 107(1982), Molecular & General Genetics, 168, 111 (1979)].

When an animal cell is used as a host, as the expression vector, anyvector can be used as long as it can exhibits its function in an animalcell, and examples thereof include pcDNA I, pcDM8 (manufactured byFunakoshi Co., Ltd.), pAGE107 [JP-A-H3-22979; Cytotechnology, 3, 133(1990)], pAS3-3 (JP-A-H2-227075), pcDM8 [Nature, 329, 840 (1987)], pcDNAI/Amp (manufactured by Invitrogen, Inc.), pcDNA3.1 (manufactured byInvitrogen, Inc.), pREP4 (manufactured by Invitrogen, Inc.), pAGE103 [J.Biochemistry, 101, 1307 (1987)], pAGE210, pME18SFL3, N5KG4PE R409K (WO2006/033386), pKANTEX93 (WO 97/10354), and the like.

As the promoter, any promoter can be used as long as it can exhibit itsfunctions in an animal cell, and examples thereof include acytomegalovirus (CMV) immediate early (IE) gene promoter, an SV40 earlypromoter, a retrovirus promoter, a metallothionein promoter, aheat-shock promoter, an SRα promoter, or a Moloney murine leukemia viruspromoter or enhancer. In addition, a human CMV IE gene enhancer may beused together with the promoter.

Examples of the host cell include a human leukemia cell Namalwa cell, amonkey cell COS cell, a Chinese hamster ovary cell CHO cell (Journal ofExperimental Medicine, 108, 945 (1958); Proc. Natl. Acad. Sci. USA, 60,1275 (1968); Genetics, 55, 513 (1968); Chromosoma, 41, 129 (1973);Methods in Cell Science, 18, 115 (1996); Radiation Research, 148, 260(1997); Proc. Natl. Acad. Sci. USA, 77, 4216 (1980); Proc. Natl. Acad.Sci. USA, 60, 1275 (1968); Cell, 6, 121 (1975); Molecular Cellgenetics,Appendix I, II (pp. 883-900), CHO/DG44, CHO-K1 (ATCC No. CCL-61),DUkXB11 (ATCC No. CCL-9096), Pro-5 (ATCC No. CCL-1781), CHO—S(LifeTechnologies, Cat #11619), Pro-3, a rat myeloma cellYB2/3HL.P2.G11.16Ag.20 (or also referred to as YB2/0), a mouse myelomacell NSO, a mouse myeloma cell 5P2/0-Ag14, a Syrian hamster cell BHK,HBT5637 (JP-A-563-000299), and the like.

As a method for introducing a recombinant vector into a host cell, anymethod can be used as long as it is a method for introducing a DNA intoan animal cell. Examples thereof include an electroporation method[Cytotechnology, 3, 133 (1990)], a calcium phosphate method(JP-A-H2-227075), a lipofection method [Proc. Natl. Acad. Sci. USA, 84,7413 (1987)], and the like.

BMP10 can be produced by culturing a microorganism having a recombinantvector incorporating a DNA encoding BMP10, or a transformant derivedfrom an animal cell or the like obtained as described above in a culturemedium, producing and accumulating the BMP10 in a culture, and thencollecting it from the culture. A method for culturing the transformantin a culture medium can be carried out according to a usual method usedfor culturing a host.

In the case where expression is carried out in a cell derived from aeukaryote, it is possible to obtain BMP10 to which a sugar or a sugarchain is added.

When culturing a microorganism transformed with a recombinant vectorusing an inducible promoter, an inducer may be added to a culture mediumas needed. For example, when a microorganism transformed with arecombinant vector using a lac promoter is cultured,isopropyl-β-D-thiogalactopyranoside or the like may be added to aculture medium, and when a microorganism transformed with a recombinantvector using a trp promoter is cultured, indoleacrylic acid or the likemay be added to a culture medium.

Examples of the culture medium in which the transformant obtained usingan animal cell as a host is cultured include RPMI 1640 medium [TheJournal of the American Medical Association, 199, 519 (1967)], Eagle'sMEM medium [Science, 122, 501 (1952)], Dulbecco's modified MEM medium[Virology, 8, 396 (1959)], Medium 199 [Proc. Soc. Exp. Biol. Med., 73, 1(1950)], Iscove's modified Dulbecco's medium (IMDM), which are generallyused, or a culture medium in which fetal bovine serum (FBS) or the likeis added to any of these culture media, and the like. The culture isusually carried out for 1 to 7 days under the conditions of pH 6 to 8and 30 to 40° C. in the presence of 5% CO₂. In addition, during theculture, an antibiotic such as kanamycin or penicillin may be added tothe culture medium as needed.

As a method for expressing a gene encoding BMP10, a method of secretoryproduction, fused protein expression, or the like [Molecular Cloning, ALaboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press(1989)] can be used in addition to direct expression.

Examples of a method for producing BMP10 include a method for producingit in a host cell, a method for secreting it out of a host cell, and amethod for producing it on an outer membrane of a host cell, and anappropriate method can be selected by changing a host cell to be used orthe structure of BMP10 to be produced.

When BMP10 is produced in a host cell or on an outer membrane of a hostcell, BMP10 can be actively secreted out of the host cell using themethod of Paulson et al. [J. Biol. Chem., 264, 17619 (1989)], the methodof Lowe et al. [Proc. Natl. Acad. Sci., USA, 86, 8227 (1989), GenesDevelop., 4, 1288 (1990)], or a method described in JP-A-H05-336963, WO94/23021, or the like.

In addition, the production amount of BMP10 can also be increased byutilizing a gene amplification system using a dihydrofolate reductasegene or the like (JP-A-H2-227075).

The obtained BMP10 can be isolated and purified, for example, asfollows.

When BMP10 is expressed in cells in a dissolved state, the cells arecollected by centrifugation after completion of the culture, suspendedin an aqueous buffer solution, followed by homogenization of the cellsusing an ultrasonic homogenizer, a French press, a Manton Gaulinhomogenizer, a Dyno mill, or the like, whereby a cell-free extractsolution is obtained.

It is possible to obtain a purified preparation from a supernatantobtained by centrifugation of the cell-free extract solution usingmethods such as usual protein isolation and purification methods, thatis, a solvent extraction method, a salting-out method using ammoniumsulfate or the like, a desalting method, a precipitation method using anorganic solvent, anion exchange chromatography using a resin such asdiethylaminoethyl (DEAE)-Sepharose or DIAION HPA-75 (manufactured byMitsubishi Chemical Corporation), cation exchange chromatography using aresin such as S-Sepharose FF (manufactured by Pharmacia Corporation),hydrophobic chromatography using a resin such as Butyl Sepharose orPhenyl Sepharose, a gel filtration method using a molecular sieve,affinity chromatography, a chromatofocusing method, electrophoresis suchisoelectric focusing electrophoresis, and the like alone or incombination.

When BMP10 is expressed in cells by forming an insoluble body, the cellsare collected and then homogenized in the same manner as describedabove, followed by centrifugation, whereby the insoluble body of theBMP10 is collected as a precipitated fraction. The collected insolublebody of the BMP10 is solubilized with a protein denaturing agent. TheBMP10 is returned to a normal conformation by diluting or dialyzing thesolubilized solution, and thereafter, a purified preparation of apolypeptide can be obtained by the same isolation and purificationmethods as described above.

When BMP10, or a derivative such as a sugar-modified body thereof isextracellularly secreted, the BMP10, or the derivative such as asugar-modified body thereof can be collected in a culture supernatant.The culture is subjected to a treatment using a method such ascentrifugation in the same manner as described above, thereby obtaininga soluble fraction, and then by using the same isolation andpurification methods as described above, a purified preparation can beobtained from the soluble fraction.

In addition, BMP10 used in the present invention can also be producedusing a chemical synthesis method such an Fmoc method or a tBoc method.Further, chemical synthesis can also be carried out using a peptidesynthesizer manufactured by Advanced Chemtech, Inc., PerkinElmer, Inc.,Pharmacia Corporation, Protein Technology Instrument, Inc.,Synthecell-Vega Biomolecules Corporation, Perceptive, Inc., ShimadzuCorporation, or the like.

(2) Immunization of Animal and Preparation of Antibody-Producing Cellsfor Fusion

An animal such as a mouse, a rat, or a hamster at 3 to 20 weeks of ageis immunized with the antigen obtained in (1), and antibody-producingcells in the spleen, the lymph node, or the peripheral blood of theanimal are collected. In addition, when a sufficient increase in theantibody titer is not observed in the above-mentioned animal due to lowimmunogenicity, a BMP9 knockout mouse can also be used as an animal tobe immunized.

The immunization is carried out by subcutaneously, intradermally,intravenously, or intraperitoneally administering an antigen to ananimal, for example, together with an appropriate adjuvant such as aFreund's complete adjuvant, an aluminum hydroxide gel, Bordetellapertussis vaccine, or the like. When the antigen is a partial peptide, aconjugate of the antigen with a carrier protein such as BSA (bovineserum albumin) or KLH (Keyhole Limpet hemocyanin) is produced and usedas an immunogen.

The administration of the antigen is carried out 2 to 10 times every 1to 2 weeks after the first administration. On day 3 to 7 after eachadministration, the blood is collected from a venous plexus of thefundus, and the antibody titer of the serum thereof is measured using anenzyme immunoassay method [Antibodies—A Laboratory Manual, Cold SpringHarbor Laboratory (1988)] or the like. An animal whose serum shows asufficient antibody titer against the antigen used for the immunizationis used as a supply source for the antibody-producing cells for fusion.

On day 3 to 7 after the final administration of the antigen, a tissuecontaining the antibody-producing cells such as the spleen is extractedfrom the immunized animal, and the antibody-producing cells arecollected. When spleen cells are used, the spleen is shredded andloosened, followed by centrifugation, and then red blood cells areremoved, whereby the antibody-producing cells for fusion are obtained.

(3) Preparation of Myeloma Cells

As myeloma cells, an established cell line obtained from a mouse isused, for example, an 8-azaguanine resistant mouse (BALB/c derived)myeloma cell line P3-X63Ag8-U1 (P3-U1) [Current Topics in Microbiologyand Immunology, 18, 1 (1978)], P3-NS1/1-Ag41 (NS-1) [European J.Immunology, 6, 511 (1976)], SP2/0-Ag14 (SP-2) [Nature, 276, 269 (1978)],P3-X63-Ag8653 (653) [J. Immunology, 123, 1548 (1979)], P3-X63-Ag8 (X63)[Nature, 256, 495 (1975)], or the like is used.

The myeloma cells are subcultured in a normal culture medium [RPM1-1640medium supplemented with glutamine, 2-mercaptoethanol, gentamicin, FBS,and 8-azaguanine], and subcultured in a normal culture medium 3 to 4days before cell fusion, and 2×10⁷ or more cells are ensured on the dayof performing the fusion.

(4) Cell Fusion and Preparation of Monoclonal Antibody-ProducingHybridoma

The antibody-producing cells for fusion obtained in (2) and the myelomacells obtained in (3) are washed well with Minimum Essential Medium(MEM) or PBS (1.83 g of disodium phosphate, 0.21 g of monopotassiumphosphate, 7.65 g of sodium chloride, 1 L of distilled water, pH 7.2),and mixed so that the cell count becomes as follows: theantibody-producing cells for fusion: the myeloma cells=5:1 to 10:1,followed by centrifugation, and then, the supernatant is removed.

After the precipitated cell aggregate is well loosened, a mixed solutionof polyethylene glycol 1000 (PEG-1000), MEM medium, anddimethylsulfoxide is added thereto while stirring at 37° C. Further, 1to 2 mL of MEM medium is added thereto several times every 1 to 2minutes, and then MEM medium is added thereto so that the total amountbecomes 50 mL. After centrifugation, the supernatant is removed. Theprecipitated cell aggregate is gently loosened, and then the cells aregently suspended in HAT medium [a normal culture medium supplementedwith hypoxanthine, thymidine, and aminopterin] as the antibody-producingcells for fusion. The resulting suspension is cultured in a 5% CO₂incubator at 37° C. for 7 to 14 days.

After the culture, a portion of the culture supernatant is withdrawn,and a cell aggregate that reacts with an antigen including BMP10, butdoes not react with an antigen not including BMP10 is selected by ahybridoma selection method such as the below-mentioned binding assay.Subsequently, cloning is repeated twice by a limiting dilution method[HT medium (a medium obtained by removing aminopterin from HAT medium)is used in the first cloning, and the normal culture medium is used inthe second cloning], and a cell in which a high antibody titer is stablyobserved is selected as a monoclonal antibody-producing hybridoma.

(5) Preparation of Purified Monoclonal Antibody

The monoclonal antibody-producing hybridoma obtained in (4) isintraperitoneally injected into a mouse or a nude mouse at 8 to 10 weeksof age having been subjected to a pristane treatment [0.5 mL of2,6,10,14-tetramethylpentadecane (pristane) is intraperitoneallyadministered, followed by rearing the mouse for 2 weeks]. In 10 to 21days, the hybridoma is converted into an ascites tumor. The ascites iscollected from this mouse, followed by centrifugation to remove solids,and then salting out is carried out with 40% to 50% ammonium sulfate.Thereafter, purification is carried out by a caprylic acid precipitationmethod, a DEAE-Sepharose column, a protein A column, or a gel filtrationcolumn, and then an IgG or IgM fraction is collected and a purifiedmonoclonal antibody is prepared.

Further, after culturing the monoclonal antibody-producing hybridomaobtained in (4) in RPMI 1640 medium supplemented with 10% FBS, or thelike, the supernatant is removed by centrifugation, and the residue issuspended in Hybridoma-SFM medium, and then cultured for 3 to 7 days.The obtained cell suspension is centrifuged, and purification is carriedout from the obtained supernatant by a protein A column or a protein Gcolumn, and then an IgG fraction is collected, whereby a purifiedmonoclonal antibody can also be obtained. Note that it is also possibleto add 5% Daigo's GF21 to the Hybridoma-SFM medium.

The determination of the subclass of the antibody is carried out by anenzyme immunoassay method using a subclass typing kit. The quantitativedetermination of a protein content can be carried out by a Lowry methodor by calculation from an absorbance at 280 nm.

(6) Selection of Monoclonal Antibody

The selection of a monoclonal antibody is carried out by a binding assayusing an enzyme immunoassay method and a kinetics analysis using Biacoredescribed below.

(6-a) Binding Assay

As an antigen, a transgenic cell obtained by introducing the expressionvector containing a cDNA encoding BMP10 obtained in (1) into E. coli,yeast, an insect cell, an animal cell, or the like, a recombinantprotein, or a purified polypeptide or a partial peptide obtained from ahuman tissue, or the like is used. When the antigen is a partialpeptide, a conjugate thereof with a carrier protein such as BSA or KLHis produced and may be used.

The antigen is dispensed in a plate such as a 96-well plate andimmobilized thereon, and thereafter, a test substance such as serum, aculture supernatant of a hybridoma, or a purified monoclonal antibody isdispensed therein as a first antibody and reacted therewith. After wellwashing with PBS or PBS containing 0.05 to 0.1% Tween 20 (hereinafteralso referred to as PBST), or the like, an anti-immunoglobulin antibodylabeled with biotin, an enzyme, a chemiluminescent substance, aradioactive compound, or the like is dispensed therein as a secondantibody and reacted therewith. After well washing with PBST, a reactioncorresponding to the labeling substance of the second antibody iscarried out, and a monoclonal antibody that specifically reacts with theimmunogen is selected.

Further, the monoclonal antibody of the present invention can beobtained by adding a test antibody to the above-mentioned binding assaysystem to cause a reaction. That is, by screening an antibody with whichthe binding of the monoclonal antibody is inhibited when a test antibodyis added, it is possible to obtain a monoclonal antibody that competeswith the obtained monoclonal antibody for binding to the amino acidsequence of BMP10 or the conformation thereof.

Further, an antibody that binds to the same epitope as an epitope whichthe monoclonal antibody of the present invention recognizes can beobtained by identifying an epitope of an antibody obtained in theabove-mentioned binding assay system, producing a partial syntheticpeptide of the identified epitope, a synthetic peptide mimicking theconformation of the epitope, or the like, and then performingimmunization therewith.

(6-b) Kinetics Analysis by Biacore

By using Biacore T100, the kinetics of binding between an antigen and atest substance are measured, and the result is analyzed with an analysissoftware attached to an instrument. After fixing an anti-mouse IgGantibody to a sensor chip CMS by an amine coupling method, a testsubstance such as a hybridoma culture supernatant or a purifiedmonoclonal antibody is allowed to flow to bind an appropriate amount,further the antigen at a plurality of known concentrations is allowed toflow, and then binding and dissociation are measured.

A kinetics analysis by a 1:1 binding model is carried out with respectto the obtained data using the software attached to the instrument toobtain various parameters.

Alternatively, after fixing human BMP10 onto the sensor chip by, forexample, an amine coupling method, a purified monoclonal antibody at aplurality of known concentrations is allowed to flow, and then bindingand dissociation are measured. A kinetics analysis by a bivalent bindingmodel is carried out with respect to the obtained data using thesoftware attached to the instrument to obtain various parameters.

2. Production of Genetically Recombinant Antibody

As production examples of genetically recombinant antibodies, methodsfor producing a human chimeric antibody and a humanized antibody will bedescribed below.

(1) Construction of Expression Vector for Genetically RecombinantAntibody

An expression vector for a genetically recombinant antibody is anexpression vector for an animal cell into which DNAs encoding CH and CLof a human antibody are incorporated, and can be constructed by cloningeach of the DNAs encoding CH and CL of a human antibody into anexpression vector for an animal cell.

As a C region of a human antibody, CH and CL of an arbitrary humanantibody can be used. For example, CH of γ1 subclass and CL of κ classof a human antibody, or the like are used. As the DNA encoding CH or CLof a human antibody, a cDNA is used, but it is also possible to use achromosomal DNA composed of an exon and an intron.

As the expression vector for an animal cell, any vector can be used aslong as it can incorporate a gene encoding a C region of a humanantibody and express the gene, and for example, pAGE107 [Cytotechnol.,3, 133 (1990)], pAGE103 [J. Biochem., 101, 1307 (1987)], pHSG274 [Gene,27, 223 (1984)], pKCR [Proc. Natl. Acad. Sci. USA, 78, 1527 (1981)],pSG1bd2-4 [Cytotechnol., 4, 173 (1990)], pSE1UK1Sed1-3 [Cytotechnol.,13, 79 (1993)], or the like is used. Further, when an IgG4PE R409Kantibody is expressed, for example, N5KG4PE R409K (WO 2006/033386), orthe like can be used.

As a promoter or an enhancer of the expression vector for an animalcell, an SV40 early promoter [J. Biochem., 101, 1307 (1987)], Moloneymurine leukemia virus LTR [Biochem. Biophys. Res. Commun., 149, 960(1987)], or an immunoglobulin H chain promoter [Cell, 41, 479 (1985)] orenhancer [Cell, 33, 717 (1983)], or the like is used.

As the expression vector for a genetically recombinant antibody, anexpression vector for a genetically recombinant antibody of a type(tandem-type) in which the antibody H chain and L chain are present onthe same vector [J. Immunol. Methods, 167, 271 (1994)] can be used fromthe viewpoints of ease of construction of the expression vector for agenetically recombinant antibody, ease of introduction into an animalcell, balancing of the expression levels of the antibody H chain and Lchain in the animal cell, and the like. In addition, a type in which theantibody H chain and L chain are present on separate vectors can also beused. As the tandem-type expression vector for a genetically recombinantantibody, pKANTEX93 (WO 97/10354), pEE18 [Hybridoma, 17, 559 (1998)],N5KG4PE R409K (WO 2006/033386), or the like is used.

(2) Acquisition of cDNA Encoding V Region of Antibody Derived fromAnimal Other than Human and Analysis of Amino Acid Sequence

Acquisition of cDNAs encoding VH and VL of a non-human antibody and ananalysis of an amino acid sequence can be carried out as follows. mRNAis extracted from hybridoma cells that produce a non-human antibody, andcDNAs are synthesized. The synthesized cDNAs are each cloned into avector such as a phage or a plasmid, thereby producing a cDNA library.

A recombinant phage or a recombinant plasmid comprising a cDNA encodingVH or VL is isolated from the library using a DNA encoding a C regionpart or a V region part of a mouse antibody as a probe, respectively.The entire nucleotide sequence of the target VH or VL of the mouseantibody in the recombinant phage or the recombinant plasmid isdetermined, respectively, and the entire amino acid sequence of VH or VLis deduced from the nucleotide sequence respectively.

As an animal other than a human for preparing hybridoma cells thatproduce a non-human antibody, a mouse, a rat, a hamster, a rabbit, orthe like is used, but any animal can be used as long as it can preparehybridoma cells.

For the preparation of the total RNA from hybridoma cells, a guanidinethiocyanate-cesium trifluoroacetate method [Methods in Enzymol., 154, 3(1987)], or a kit such as RNA easy Kit (manufactured by QIAGEN, Inc.),or the like is used.

In the preparation of mRNA from the total RNA, an oligo (dT)-immobilizedcellulose column method [Molecular Cloning, A Laboratory Manual, SecondEdition, Cold Spring Harbor Laboratory Press (1989)], or a kit such asOligo-dT30 <Super> mRNA Purification Kit (manufactured by Takara BioInc.), or the like is used. Further, it is also possible to prepare mRNAfrom hybridoma cells using a kit such as Fast Track mRNA Isolation Kit(manufactured by Invitrogen, Inc.), or QuickPrep mRNA Purification Kit(manufactured by Pharmacia Corporation).

In the synthesis of cDNAs and the production of a cDNA library, a knownmethod [Molecular Cloning, A Laboratory Manual, Second Edition, ColdSpring Harbor Laboratory Press (1989), Current Protocols in MolecularBiology, Supplement 1, John Wiley & Sons (1987-1997)], or a kit such asSuperScript Plasmid System for cDNA Synthesis and Plasmid Cloning(manufactured by Invitrogen, Inc.) or ZAP-cDNA Synthesis Kit(manufactured by Stratagene Corporation), or the like is used.

When the cDNA library is produced, as the vector into which a cDNAsynthesized using mRNA extracted from hybridoma cells as a template isincorporated, any vector can be used as long as it is a vector capableof incorporating the cDNA. For example, ZAP ExPress [Strategies, 5, 58(1992)], pBluescript II SK(+) [Nucleic Acids Research, 17, 9494 (1989)],λZAPII (manufactured by Stratagene Corporation), λgt 10, λgt 11 [DNACloning: A Practical Approach, I, 49 (1985)], Lambda BlueMid(manufactured by Clontech Laboratories, Inc.), λEx Cell, pT7T3-18U(manufactured by Pharmacia Corporation), pcD2 [Mol. Cell. Biol., 3, 280(1983)], pUC18 [Gene, 33, 103 (1985)], or the like is used.

As E. coli into which a cDNA library constructed by a phage or a plasmidvector is introduced, any E. coli can be used as long as it canintroduce, express, and maintain the cDNA library. For example, XL-1BlueMRF [Strategies, 5, 81 (1992)], C600 [Genetics, 39, 440 (1954)], Y1088,Y1090 [Science, 222, 778 (1983)], NM522 [J. Mol. Biol., 166, 1 (1983)],K802 [J. Mol. Biol., 16, 118 (1966)], JM105 [Gene, 38, 275 (1985)], orthe like is used.

In the selection of a cDNA clone encoding VH or VL of a non-humanantibody from the cDNA library, a colony hybridization method using anisotope or a fluorescently labeled probe, or a plaque hybridizationmethod [Molecular Cloning, A Laboratory Manual, Second Edition, ColdSpring Harbor Laboratory Press (1989)], or the like is used.

In addition, it is possible to prepare a cDNA encoding VH or VL bypreparing a primer and performing a polymerase chain reaction method[hereinafter referred to as a PCR method, Molecular Cloning, ALaboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press(1989), Current Protocols in Molecular Biology, Supplement 1, John Wiley& Sons (1987-1997)] using a cDNA or a cDNA library synthesized from mRNAas a template.

The selected cDNA is cleaved with an appropriate restriction enzyme orthe like, and then cloned into a plasmid such as pBluescript SK(−)(manufactured by Stratagene Corporation), and the nucleotide sequence ofthe cDNA is determined by a commonly used nucleotide sequence analysismethod or the like. In the nucleotide sequence analysis method, forexample, after performing a reaction such as a dideoxy method [Proc.Natl. Acad. Sci. USA, 74, 5463 (1977)], an automatic nucleotide sequenceanalyzer such as ABI Prism 3700 (manufactured by PE Biosystems, Inc.) orA. L. F. DNA sequencer (manufactured by Pharmacia Corporation) is used.

By deducing the entire amino acid sequence of each of VH and VL from thedetermined nucleotide sequence and comparing it with the entire aminoacid sequence of each of VH and VL of a known antibody [A. L. F. DNA, USDept. Health and Human Services (1991)], it is confirmed whether theobtained cDNA encodes the complete amino acid sequence of each of VH andVL of the antibody including a secretion signal sequence.

With respect to the complete amino acid sequence of each of VH and VL ofthe antibody including a secretion signal sequence, by comparison withthe entire amino acid sequence of each of VH and VL of a known antibody[A. L. F. DNA, US Dept. Health and Human Services (1991)], the length ofthe secretion signal sequence and the N-terminal amino acid sequence canbe deduced, and further the subgroup to which these belong can beidentified. In addition, the amino acid sequence of each CDR of VH andVL can also be found out by comparison with the amino acid sequence ofeach of VH and VL of a known antibody [A. L. F. DNA, US Dept. Health andHuman Services (1991)].

Further, by using the obtained complete amino acid sequence of each ofVH and VL, it is possible to confirm whether the complete amino acidsequence of each of VH and VL is new by, for example, carrying out ahomology search by a BLAST method [J. Mol. Biol., 215, 403 (1990)] orthe like with respect to an arbitrary database such as SWISS-PROT orPIR-Protein.

(3) Construction of Human Chimeric Antibody Expression Vector

By cloning each cDNA encoding VH or VL of a non-human antibody upstreamof each gene encoding CH or CL of a human antibody in the expressionvector for a genetically recombinant antibody obtained in (1), a humanchimeric antibody expression vector can be constructed.

In order to ligate the cDNA encoding VH or VL of a non-human antibody atthe 3′ end side with CH or CL of a human antibody at the 5′ end side,cDNAs of VH and VL designed so that the nucleotide sequence of aligation region encodes an appropriate amino acid and to become anappropriate restriction enzyme recognition sequence are produced.

The produced cDNAs of VH and VL are each cloned upstream of each geneencoding CH or CL of a human antibody in the expression vector for ahuman CDR-grafted antibody obtained in (1) so that they are expressed inan appropriate form, whereby a human chimeric antibody expression vectoris constructed.

In addition, each cDNA encoding VH or VL of a non-human antibody isamplified by a PCR method using a synthetic DNA containing anappropriate restriction enzyme recognition sequence at both ends, andcan be cloned into the expression vector for a genetically recombinantantibody obtained in (1).

(4) Construction of cDNA Encoding V Region of Human CDR-Grafted Antibody

A cDNA encoding VH or VL of a human CDR-grafted antibody can beconstructed as follows.

Each amino acid sequence of FR of VH or VL of a human antibody, to whichthe amino acid sequence of a CDR of VH or VL of a non-human antibody isto be grafted is selected. As the amino acid sequence of FR to beselected, any amino acid sequence can be used as long as it is derivedfrom a human antibody.

For example, an amino acid sequence of FR of a human antibody registeredin a database such as Protein Data Bank, or a common amino acid sequencein each subgroup of FR of a human antibody [A. L. F. DNA, US Dept.Health and Human Services (1991)], or the like is used. In order tosuppress a decrease in the binding activity of an antibody, an aminoacid sequence of FR having a homology as high as possible (at least 60%or more) with the amino acid sequence of FR of VH or VL of the originalantibody is selected.

Subsequently, each of the amino acid sequences of the CDRs of theoriginal antibody is grafted to the selected amino acid sequence of FRof VH or VL of a human antibody, and each amino acid sequence of VH orVL of a human CDR-grafted antibody is designed. By converting thedesigned amino acid sequence into a DNA sequence in consideration of theusage frequency of codons found in the nucleotide sequence of theantibody gene [A. L. F. DNA, US Dept. Health and Human Services (1991)],each DNA sequence encoding the amino acid sequence of VH or VL of ahuman CDR-grafted antibody is designed.

Based on the designed DNA sequences, several synthetic DNAs having alength of around 100 nucleotides are synthesized and a PCR reaction iscarried out using them. In this case, from the viewpoint of the reactionefficiency in the PCR reaction and the length of a synthesizable DNA,preferably 6 synthetic DNAs are designed for each of the H chain and theL chain.

Further, by introducing an appropriate restriction enzyme recognitionsequence at the 5′ end of the synthetic DNA located at both ends, a cDNAencoding VH or VL of a human CDR-grafted antibody can be easily clonedinto the expression vector for a human CDR-grafted antibody obtained in(1).

Alternatively, the cloning can be carried out by using a full-lengthsynthetic DNA of each of the H chain and the L chain synthesized as asingle DNA based on the designed DNA sequences.

After the PCR reaction, each amplification product is cloned into aplasmid such as pBluescript SK(−) (manufactured by StratageneCorporation), the nucleotide sequence is determined by the same methodas the method described in (2), and a plasmid having a DNA sequenceencoding the amino acid sequence of VH or VL of a desired humanCDR-grafted antibody is obtained.

(5) Alteration of Amino Acid Sequence of V Region of Human CDR-GraftedAntibody

The antigen-binding activity of a human CDR-grafted antibody preparedmerely by grafting only CDRs of VH and VL of a non-human antibody to FRsof VH and VL of a human antibody is decreased as compared with that ofthe original non-human antibody [BIO/TECHNOLOGY, 9, 266 (1991)].

In the human CDR-grafted antibody, the lowered antigen-binding activitycan be increased by identifying an amino acid residue directly involvedin the binding to an antigen, an amino acid residue interacting with anamino acid residue of a CDR, and an amino acid residue maintaining theconformation of the antibody and indirectly involved in the binding toan antigen in the amino acid sequences of FRs of VH and VL of a humanantibody, and substituting such an amino acid residue with an amino acidresidue of the original non-human antibody.

In order to identify an amino acid residue of FR involved in theantigen-binding activity, it is possible to construct and analyze theconformation of the antibody using X-ray crystallography [J. Mol. Biol.,112, 535 (1977)], or computer modeling [Protein Engineering, 7, 1501(1994)], or the like. Further, it is possible to obtain an altered humanCDR-grafted antibody having a necessary antigen-binding activity byproducing several types of variants for each antibody, and repeatedlyexamining the correlation with the antigen-binding activity thereofthrough trial and error.

The amino acid residues of FRs of VH and VL of a human antibody can bealtered by carrying out a PCR reaction described in (4) using asynthetic DNA for alteration. With respect to the amplification productafter the PCR reaction, the nucleotide sequence is determined to confirmthat the desired alteration has been carried out by the method describedin (2).

(6) Construction of cDNA Encoding V Region of Humanized Antibody bySurface Reconstruction Method

A cDNA encoding VH or VL of a humanized antibody by a surfacereconstruction method can be constructed as follows.

In the amino acid sequence of FR of VH or VL of a non-human antibody, anamino acid residue considered to have a low effect on theantigen-binding activity is selected, respectively, and the amino acidresidue is substituted with an amino acid residue considered to havelower antigenicity than the amino acid residue.

The thus designed amino acid sequence of VH or VL is converted into aDNA sequence in consideration of the usage frequency of codons found inthe nucleotide sequence of the antibody gene [A. L. F. DNA, US Dept.Health and Human Services (1991)], and each DNA sequence encoding theamino acid sequence of VH or VL of a humanized antibody by a surfacereconstruction method is designed, respectively.

Based on the designed DNA sequences, several synthetic DNAs having alength of around 100 nucleotides are synthesized and a PCR reaction iscarried out using them. In this case, from the viewpoint of the reactionefficiency in the PCR reaction and the length of a synthesizable DNA,preferably 6 synthetic DNAs are designed for each of the H chain and theL chain.

Further, by introducing an appropriate restriction enzyme recognitionsequence at the 5′ end of the synthetic DNA located at both ends, a cDNAencoding VH or VL of a humanized antibody by a surface reconstructionmethod can be easily cloned into the expression vector for a humanizedantibody obtained in (1).

Alternatively, the cloning can be carried out by using a full-lengthsynthetic DNA of each of the H chain and the L chain synthesized as asingle DNA based on the designed DNA sequences.

After the PCR reaction, each amplification product is cloned into aplasmid such as pBluescript SK(−) (manufactured by StratageneCorporation), the nucleotide sequence is determined by the same methodas the method described in (2), and a plasmid having a DNA sequenceencoding the amino acid sequence of VH or VL of a desired human antibodyis obtained.

(7) Construction of Humanized Antibody Expression Vector

A humanized antibody expression vector can be constructed by cloningeach cDNA encoding VH or VL of a constructed genetically recombinantantibody upstream of each gene encoding CH or CL of a human antibody ofthe expression vector for a genetically recombinant antibody obtained in(1).

For example, the cloning is carried out upstream of each gene encodingCH or CL of a human antibody in the expression vector for a humanizedantibody obtained in (1) by introducing an appropriate restrictionenzyme recognition sequence at the 5′ end of the synthetic DNA locatedat both ends among the synthetic DNAs used when constructing VH or VL ofthe humanized antibody obtained in (4), (5), or (6) so that they areexpressed in an appropriate form.

(8) Transient Expression of Genetically Recombinant Antibody

By transiently expressing a genetically recombinant antibody using thegenetically recombinant antibody expression vector obtained in (3) and(7), or an expression vector obtained by alteration thereof, theantigen-binding activities of many types of humanized antibodiesproduced can be efficiently evaluated.

As a host cell into which the expression vector is introduced, any cellcan be used as long as it is a host cell capable of expressing agenetically recombinant antibody. For example, a COS-7 cell (ATCC No:CRL 1651) is used [Methods in Nucleic Acids Res., CRC Press, 283(1991)].

In the introduction of the expression vector into a COS-7 cell, aDEAE-dextran method [Methods in Nucleic Acids Res., CRC Press (1991)], alipofection method [Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)], or thelike is used.

After the introduction of the expression vector, the expression leveland the antigen-binding activity of the genetically recombinant antibodyin a culture supernatant are measured using an enzyme immunoassay method[Monoclonal Antibodies-Principles and practice, Third Edition, AcademicPress (1996), Antibodies-A Laboratory Manual, Cold Spring HarborLaboratory (1988), Monoclonal Antibody Experimental Manual, Kodanshascientific books (1987)], or the like.

(9) Acquisition of Transformant Stably Expressing GeneticallyRecombinant Antibody and Preparation of Genetically Recombinant Antibody

A transformant that stably expresses a genetically recombinant antibodycan be obtained by introducing the genetically recombinant antibodyexpression vector obtained in (3) and (7) into an appropriate host cell.

In the introduction of the expression vector into a host cell, anelectroporation method [JP-A-H2-257891, Cytotechnology, 3, 133 (1990)],or the like is used.

As a host cell into which the genetically recombinant antibodyexpression vector is introduced, any cell can be used as long as it is ahost cell capable of expressing a genetically recombinant antibody.

For example, CHO-K1 (ATCC No. CCL-61), DUkXB11 (ATCC No. CCL-9096),Pro-5 (ATCC No. CCL-1781), CHO—S(Life Technologies, Cat #11619), ratmyeloma cells YB2/3HL.P2.G11.16Ag.20 (or also referred to as YB2/0),mouse myeloma cells NSO, mouse myeloma cells SP2/0-Ag14 (ATCC No. CRL1581), mouse P3-X63-Ag8653 cells (ATCC No. CRL 1580), dihydrofolatereductase gene-deficient CHO cells [Proc. Natl. Acad. Sci. USA, 77, 4216(1980)], Lec13 having acquired lectin resistance [Somatic Cell andMolecular Genetics, 12, 55 (1986)], α1,6-fucosyltransferasegene-deficient CHO cells (WO 2005/035586, WO 02/31140), RatYB2/3HL.P2.G11.16Ag.20 cells (ATCC No: CRL 1662), or the like is used.

After introduction of the expression vector, a transformant that stablyexpresses a genetically recombinant antibody is selected by culturingthe transformant in a culture medium for animal cell culture containingan agent such as G418 sulfate (JP-A-H2-257891).

As the culture medium for animal cell culture, RPMI 1640 medium(manufactured by Invitrogen, Inc.), GIT medium (manufactured by NipponPharmaceutical Co., Ltd.), EX-CELL 301 medium (manufactured by JRHBiosciences, Inc.), IMDM medium (manufactured by Invitrogen, Inc.),Hybridoma-SFM medium (manufactured by Invitrogen, Inc.), or a medium inwhich any of various additives such as FBS is added to any of thesemedia, or the like is used.

By culturing the obtained transformant in a culture medium, agenetically recombinant antibody is expressed and accumulated in theculture supernatant. The expression level and the antigen-bindingactivity of the genetically recombinant antibody in the culturesupernatant can be measured by an ELISA method or the like. In addition,the transformant can increase the expression level of the geneticallyrecombinant antibody utilizing a DHFR amplification system(JP-A-H2-257891) or the like.

The genetically recombinant antibody is purified from the culturesupernatant of the transformant using a protein A column [MonoclonalAntibodies—Principles and Practice, Third Edition, Academic Press(1996), Antibodies—A Laboratory Manual, Cold Spring Harbor Laboratory(1988)]. In addition, it is also possible to combine methods used forpurifying a protein such as gel filtration, ion exchange chromatography,and ultrafiltration.

The molecular weights of an H chain, an L chain, or the entire antibodymolecule of a purified genetically recombinant antibody can be measuredusing polyacrylamide gel electrophoresis [Nature, 227, 680 (1970)], or aWestern blotting method [Monoclonal Antibodies—Principles and Practice,Third Edition, Academic Press (1996), Antibodies—A Laboratory Manual,Cold Spring Harbor Laboratory (1988)], or the like.

3. Evaluation of Activity of Purified Monoclonal Antibody or AntibodyFragment Thereof

The evaluation of the activity of the purified monoclonal antibody orthe antibody fragment thereof of the present invention can be carriedout as follows.

The binding activity to BMP10 and BMP10 expressing tissue is measuredusing a binding assay described in the above 1-(6-a) and a surfaceplasmon resonance method using a Biacore system or the like described inthe above (6-b). Further, it can be measured using a fluorescentantibody method [Cancer Immunol. Immunother., 36, 373 (1993)] or thelike.

The production of a bispecific antibody can be carried out by, forexample, a known method such as a method described in WO 2009/131239.

4. Therapeutic Method for Disease Using Anti-BMP10 Monoclonal Antibodyor Antibody Fragment Thereof of the Present Invention

The monoclonal antibody or the antibody fragment thereof of the presentinvention can be used for a treatment of hypertension and a hypertensivedisease.

A therapeutic agent comprising the monoclonal antibody or the antibodyfragment thereof of the present invention or a derivative thereof maycontain only the antibody or the antibody fragment thereof, or aderivative thereof as an active ingredient, however, in general, it isprovided as a pharmaceutical preparation produced by mixing it togetherwith one or more pharmacologically acceptable carriers using a methodknown in the technical field of pharmaceutics.

Examples of a route of administration include oral administration orparenteral administration such as intraoral, intra-airway, intrarectal,subcutaneous, intramuscular, or intravenous administration. Examples ofa dosage form include a spray, a capsule, a tablet, a powder, a granule,a syrup, an emulsion, a suppository, an injection, an ointment, a tape,and the like.

Examples of the pharmaceutical preparation suitable for oraladministration include an emulsion, a syrup, a capsule, a tablet, apowder, a granule, and the like.

A liquid preparation such as an emulsion or a syrup is produced usingwater, a saccharide such as sucrose, sorbitol, or fructose, a glycolsuch as polyethylene glycol or propylene glycol, an oil such as sesameoil, olive oil, or soybean oil, a preservative such as ap-hydroxybenzoic acid ester, a flavor such as strawberry flavor orpeppermint, or the like, as an additive.

A capsule, a tablet, a powder, a granule, or the like can be producedusing an excipient such as lactose, glucose, sucrose, or mannitol, adisintegrating agent such as starch or sodium alginate, a lubricant suchas magnesium stearate or talc, a binder such as polyvinyl alcohol,hydroxypropyl cellulose, or gelatin, a surfactant such as a fatty acidester, a plasticizer such as glycerin, or the like as an additive.

Examples of the pharmaceutical preparation suitable for parenteraladministration include an injection, a suppository, a spray, and thelike.

An injection is produced using a carrier composed of a salt solution, aglucose solution, or a mixture of both, or the like.

A suppository is produced using a carrier such as cacao butter, ahydrogenated fat, or carboxylic acid.

A spray is produced using a carrier which does not stimulate the buccaland airway mucous membranes of a recipient and disperses the monoclonalantibody or the antibody fragment thereof of the present invention asfine particles so as to facilitate absorption thereof, or the like. Asthe carrier, for example, lactose, glycerin, or the like is used. Inaddition, it can also be produced as an aerosol or a dry powder.

Further, a component exemplified as the additive for the pharmaceuticalpreparation suitable for oral administration can also be added to theabove-mentioned parenteral preparation.

Hereinafter, the present invention will be more specifically describedby way of Examples, however, the present invention is not limited to thefollowing Examples.

Reagents to be used shall be used according to the package insert unlessotherwise particularly stated.

EXAMPLES [Example 1] Preparation of Anti-Human BMP10 Monoclonal Antibody1-1) Preparation of Immunogen

As an immunogen, a human BMP10 recombinant protein or a human BMP10mature dimer (manufactured by R & D Systems, Inc., Cat #2926-BP) wasused. The human BMP10 recombinant protein was prepared according to themethod described in Example 20 in WO 2014/007198. The human BMP10recombinant protein prepared by this method includes a mature protein,an N-terminal propeptide protein, and a full-length protein.

1-2) Immunization of Animal and Preparation of Antibody-Producing Cells

An antigen suspension containing the human BMP10 prepared in Example 1,1-1) as an antigen was prepared according to the package insert usingSigma Adjuvant System (registered trademark) (manufactured bySigma-Aldrich Co. LLC) or Alum+pertussis vaccine adjuvant (manufacturedby Nacalai Tesque, Inc.) as an adjuvant, and thereafter, a WKY/NcrlCrljrat or an SD rat was immunized therewith through intraperitoneal andsubcutaneous or intramuscular routes. The amount of the antigen used forthe immunization was set to 20 μg/head in the case of the human BMP10recombinant protein, and 10 μg/head in the case of the human BMP10mature dimer. The immunization was carried out twice or four times intotal including the final boosting. The spleen was excised 3 to 4 daysafter the final administration.

The excised spleen was shredded in Minimum Essential Media (manufacturedby Nacalai Tesque, Inc.) (hereinafter referred to as MEM medium), andspleen cells were collected by centrifugation (1200 rpm, 5 minutes).Since the obtained spleen cell fraction contains red blood cells, thered blood cells were removed by adding RED Blood Cell Lysing Buffer(manufactured by Sigma-Aldrich Co. LLC) and treated on ice.Alternatively, the iliac lymph node was collected and cells wereloosened in MEM medium to obtain lymphocytes. The obtained spleen cellsor lymphocytes were washed twice with MEM medium, and then provided forcell fusion.

1-3) Preparation of Mouse Myeloma Cells

An 8-azaguanine resistant mouse myeloma cell line P3-U1 [P3X63Ag8U.1,ATCC: CRL-1597 European Journal of Immunology, 6, 511 (1976)] wassubjected to conditioned culture in a medium obtained by addinggentamycin (10 μg/mL) to S-Clone Cloning Medium CM-B (manufactured bySanko Junyaku Co., Ltd.) (hereinafter referred to as a serum-freeculture medium) so as to ensure a necessary cell count (4×10⁷ cells ormore) at the time of cell fusion, and provided for cell fusion.

1-4) Production of Hybridoma

The mouse spleen cells or the lymphocytes obtained in Example 1, 1-2)and the myeloma cells obtained in Example 1, 1-3) were mixed at 8:1,followed by centrifugation (1200 rpm, 5 min). To the obtainedprecipitated fraction (cell aggregate), 500 μL of a mixed liquid ofpolyethylene glycol 1000 (manufactured by Junsei Chemical Co., Ltd., Cat#69257-1210), MEM medium, and dimethyl sulfoxide (DMSO, manufactured bySigma-Aldrich Co. LLC, Cat #D2650) was gradually added while gentlyshaking. Subsequently, 5 mL of MEM medium was added to the cell liquidwhile gently shaking, and further 45 mL of MEM medium was added thereto.Then, a tube containing the cell liquid was centrifuged (900 rpm, 5min).

The obtained precipitated fraction (cell aggregate) was seeded into a96-well plate using a serum-free culture medium containing HAT at 200μL/well excluding the row A. At that time, the spleen cell count or thelymphocyte count was adjusted to 1.5×10⁷ cells/18 mL per plate, and thecells were cultured under the condition of 37° C. and 5% CO₂. Mediumexchange was appropriately carried out using a serum-free culture mediumcontaining HAT until the cells in the well reached a cell count suitablefor screening.

1-5) Screening of Anti-BMP10 Antibody-Producing Hybridoma by Solid-PhaseAntigen ELISA

In the screening of an anti-BMP10 antibody-producing hybridoma, asolid-phase antigen ELISA system in which human BMP10 was immobilized onan ELISA plate was used. Specifically, a solution in which the humanBMP10 recombinant prepared in Example 1, 1-1) or a human BMP10 maturedimer (manufactured by R & D Systems, Inc., Cat #2926-BP) was preparedat 0.5 μg/mL with a phosphate buffer solution (manufactured by NacalaiTesque, Inc.) was dispensed in a 96-well ELISA plate (F96 MAXISORPNUNC-IMMNO PLATE, manufactured by Thermo Fisher Scientific, Inc., Cat#442404) at 50 μL/well, and adsorption was carried out by leaving theplate to stand overnight at 4° C.

After the immobilization solution was removed, washing was carried out 3to 5 times with PBS, and 1% BSA-PBS (manufactured by Nacalai Tesque,Inc., Cat #099968-35) was added at 200 μL/well, and blocking was carriedout by leaving the plate to stand at room temperature for 1 hour.

Subsequently, the hybridoma supernatant was dispensed at 50 μL/well, andthe plate was left to stand at room temperature for 1 hour. After thisplate was washed 3 to 5 times with PBST, Goat F(ab′)₂ Anti-Rat IgG-Fc(HRP), pre-adsorbed (manufactured by Abcam PLC, Cat #ab6257) diluted1000 times with 1% BSA-PBS was dispensed at 50 μL/well, and the platewas left to stand at room temperature for 1 hour.

This plate was washed 3 to 5 times with PBST, an ABTS(2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic Acid, manufactured byWako, Cat #016-08521) substrate solution or a TMB substrate solution wasadded at 50 μL/well so as to develop a color. When appropriate colordevelopment was obtained, a 5% SDS solution or a 1 mol/L hydrochloricacid was added at 50 μL/well, and an absorbance (415 nm to 490 nm) at asample wavelength of 415 nm and a reference wavelength of 490 nm, or anabsorbance (450 nm to 570 nm) at a sample wavelength of 450 nm and areference wavelength of 570 nm was measured using a plate reader(Spectra Max, manufactured by Molecular Devices, LLC).

1-6) Preparation of Cell Capable of Detecting BMP Signal

In the screening of a hybridoma that produces an anti-BMP10 antibody,cells capable of detecting a signal of various types of BMP proteins(hereinafter, BMP signal detection cells) were used. As the BMP signaldetection cells, Id1-Luc/CHO cells described in Example 5 ofJP-A-2017-25011 were used.

1-7) Screening of Anti-BMP10 Neutralizing Antibody-Producing HybridomaUsing Id1-Luc/CHO

To a 96-well fluorescence and luminescence plate (manufactured byCorning, Inc., Cat #3916), a human BMP10 mature dimer was added at afinal concentration of 3 ng/mL. Subsequently, a hybridoma culturesupernatant was added at a final concentration of 50%. Thereafter, anId1-Luc/CHO cell liquid in which the cells were suspended in Excell 325medium [Excell 325 PF CHO (manufactured by SAFC, Inc., Cat #14340C-1000mL), 4 mM L-glutamine, 1×Penicillin, 1× Streptomycin (manufactured byNacalai, Inc., Cat #09367-34), 0.5 μg/mL hygromycin] was added at 5×10⁴cells/well.

Note that all samples were diluted with Excell 325 medium so as toachieve 100 μL/well when the samples were added. Thereafter, the liquidin the well was made uniform with a plate mixer, and the cells werecultured at 37° C. for 20 hours. After 20 hours, an assay solution ofNano-Glo Luciferase Assay (manufactured by Promega Corporation, Cat#N1120) prepared according to the package insert was added at 40μL/well, followed by stirring, and then, a luciferase activity wasmeasured using Glomax (manufactured by Promega Corporation).

The neutralizing activity (%) of the antibody in the hybridoma culturesupernatant was calculated by setting the value of the well in whichonly the BMP10 mature dimer was added without adding the hybridomaculture supernatant to be 0%, and the value of the well in which onlyExcell 325 medium was added without adding an antibody to be 100%.

1-8) Isolation of Anti-BMP10 Neutralizing Antibody-Producing Hybridoma

A hybridoma that exhibits a neutralizing activity of 50% or more in theabove-mentioned screening was determined to be positive. The hybridomadetermined to be positive was monocloned by limiting dilution with aserum-free culture medium, and seeding into a 96-well plate. Themonocloning was carried out once to twice in total with respect to thehybridoma derived from the well determined to be positive at the firsttime. By the above operation, hybridomas that produce 18C1 antibody,12H3 antibody, and 11H10 antibody, respectively, were isolated.

1-9) Large-Scale Acquisition of Antibody from Hybridoma

Each of the hybridomas isolated in Example 1, 1-8) was seeded into twolarge flask bottles. A serum-free culture medium was used as the culturemedium. After culturing at 37° C. for 6 to 8 days, the culture mediumcontaining the cells was collected. The collected culture medium wascentrifuged, and the obtained culture supernatant was filtered through a0.22 μm filter.

The anti-human BMP10 antibody was purified from the culture supernatantfiltered through the filter using an open column packed with Protein GSepharose 4 Fast Flow (manufactured by GE Healthcare, Inc.) orAb-Capchure Extra (manufactured by Protenova Co., Ltd.).

[Example 2] Comparison of BMP10 Neutralizing Activity Between ObtainedAntibody and Known Antibody Using Id1-Luc/CHO Cells

Comparison of the BMP10 neutralizing activity with respect to theobtained anti-BMP10 antibodies and a known antibody was carried outusing the Id1-Luc/CHO cells produced in Example 1, 1-6). To a 96-wellfluorescence and luminescence plate (manufactured by Corning, Inc., Cat#3916), a human BMP10 mature dimer (manufactured by R & D Systems, Inc.,Cat #2926-BP) was added at a final concentration of 3 ng/mL.Subsequently, a known antibody MAB2926 (the known antibody refers toMAB2926), and the 18C1 antibody, the 12H3 antibody, and the 11H10antibody, each of which is an anti-BMP10 antibody purified in Example 1,1-9), or a control antibody (Purified Rat IgG1 λ Isotype control,manufactured by BD, Inc., Cat #553993) were prepared at 6 concentrationsby 3-fold serial dilution from the final concentration of 3000 ng/mL,and then added.

Thereafter, an Id1-Luc/CHO cell liquid in which the cells were suspendedin Excell 325 medium [Excell 325 PF CHO (manufactured by SAFC, Inc., Cat#14340C-1000 mL), 4 mM L-glutamine, 1×Penicillin, 1×Streptomycin(manufactured by Nacalai, Inc., Cat #09367-34), 0.5 mg/mL hygromycin]was added at 5×10⁴ cells/well. After all samples were added, the liquidin each well was made uniform with a plate mixer, and the cells werecultured at 37° C. for 20 hours.

After 20 hours, an assay solution of Nano-Glo Luciferase Assay(manufactured by Promega Corporation, Cat #N1120) prepared according tothe package insert was added at 40 μL/well, followed by stirring, andthen, a luciferase activity was measured using Glomax (manufactured byPromega Corporation). The neutralizing activity (%) of the antibody wascalculated by setting the value of the well in which only the BMP10mature dimer was added without adding an antibody to be 0%, and thevalue of the well in which only Excell 325 medium was added withoutadding an antibody to be 100%. The results are shown in FIG. 1.

As shown in FIG. 1, any antibody exhibited a neutralizing activityagainst BMP10 except for the control antibody. It was found that theknown antibody MAB2926 cannot completely neutralize 3 ng/mL of matureBMP10 at 3 μg/mL. On the other hand, the obtained antibodies: the 11H10antibody, the 12H3 antibody, and the 18C1 antibody could completelyneutralize 3 ng/mL of mature BMP10 at 3 μg/mL. Further, the 18C1antibody and the 12H3 antibody exhibited a BMP10 neutralizing activityat a lower concentration than MAB2926.

From the above results, it was revealed that all the obtained threeantibodies are antibodies having a significantly improved BMP10neutralizing activity as compared with the known antibody.

[Example 3] Comparison of BMP10 Neutralizing Activity Between ObtainedAntibody and Known Antibody Using Human ALK1 Expressing Reporter Cells

The neutralizing activities against BMP10 of the obtained anti-BMP10antibodies and a known antibody were compared using human ALK1expressing reporter cells made to highly express human ALK1. As theknown antibody, MAB2926 (R & D Systems, Inc.) was used.

3-1) Production of Human ALK1 Expressing Reporter Cells

As the human ALK1 expressing reporter cells, ALK1/Id1-Luc/CHO cellsdescribed in Example 6 in JP-A-2017-25011 were used.

3-2) Comparison of BMP10 Neutralizing Activity Between Newly ObtainedAntibody and Known Antibody

To a 96-well fluorescence and luminescence plate (manufactured byCorning, Inc., Cat #3916), human BMP10 mature dimer (manufactured by R &D Systems, Inc., Cat #2926-BP) was added at a final concentration of 0.3ng/mL. Subsequently, the 18C1 antibody, the 12H3 antibody, the 11H10antibody or MAB2926, each of which is an anti-BMP10 antibody, or acontrol antibody (Purified Rat IgG1 λ Isotype control, manufactured byBD, Inc., Cat #553993) was prepared at 6 concentrations by 3-fold serialdilution from the final concentration of 3000 ng/mL, and then added.

Thereafter, an ALK1/Id1-Luc/CHO cell suspension prepared with Excell 325medium was added at 5×10⁴ cells/well. All samples were diluted withExcell 325 medium so as to achieve 100 μL/well when combining humanBMP10, the antibody dilution solution, and the cell suspension.Thereafter, the liquid in the well was made uniform with a plate mixer,and the cells were cultured at 37° C. for 20 hours.

After 20 hours, an assay solution of Nano-Glo Luciferase Assay preparedaccording to the package insert was added at 40 μL/well, followed bystirring, and then, a luciferase activity was measured using Glomax(manufactured by Promega Corporation). The neutralizing activity (%) ofthe antibody was calculated by setting the value of the well in whichonly the BMP10 mature dimer was added without adding an antibody to be0%, and the value of the well in which only Excell 325 medium was addedwithout adding an antibody to be 100%. The results are shown in FIG. 2.

As shown in FIG. 2, the control antibody and the known antibody MAB2926did not exhibit a neutralizing activity in the ALK1/Id1-Luc/CHO cells.On the other hand, the obtained antibodies: the 18C1 antibody, the 12H3antibody, and the 11H10 antibody exhibited a distinct BMP10 neutralizingactivity also in the ALK1/Id1-Luc/CHO cells. Further, the strength ofthe neutralizing activity was higher in the order of the 18C1 antibody,the 12H3 antibody, and the 11H10 antibody.

From the above results, it was revealed that the obtained antibodies arenovel antibodies exhibiting a neutralizing activity in ALK1 highlyexpressing cells.

[Example 4] Inhibitory Effect of Obtained Antibody on Various Types ofBMP Family Molecules

In order to confirm that the 18C1 antibody specifically neutralizesBMP10, an inhibitory effect of the 18C1 antibody on various types of BMPsignals was examined using the Id1-Luc/CHO cells produced in Example 1,1-6).

To a 96-well fluorescence and luminescence plate (manufactured byCorning, Inc., Cat #3916), each of various types of human BMP maturedimers was added at a final concentration of 3 ng/mL, and subsequently,a dilution solution of the 18C1 antibody was added at a finalconcentration of 1.0 μg/mL. As the various types of human BMP10 maturedimers, human BMP2, human BMP4, human BMP6, human BMP7, human BMP9,human BMP10, human BMP15, human GDF5, and human GDF7 (all manufacturedby R & D Systems, Inc.) were used. Thereafter, an Id1-Luc/CHO cellliquid in which the cells were suspended in Excell 325 medium [Excell325 PF CHO (manufactured by SAFC, Inc., Cat #14340C-1000 mL), 4 mML-glutamine, 1×Penicillin, 1×Streptomycin (manufactured by Nacalai,Inc., Cat #09367-34), 0.5 μg/mL hygromycin] was added at 5×10⁴cells/well.

All samples were diluted with Excell 325 medium so as to achieve 100μL/well in the end. Thereafter, the liquid in the well was made uniformwith a plate mixer, and the cells were cultured at 37° C. for 20 hours.

After 20 hours, an assay solution of Nano-Glo Luciferase Assay(manufactured by Promega Corporation, Cat #N1120) prepared according tothe package insert was added at 40 μL/well, followed by stirring, andthen, a luciferase activity was measured using Glomax (manufactured byPromega Corporation).

The neutralizing activity (%) of the antibody was calculated by settingthe value of the well in which only the BMP mature dimer was addedwithout adding an antibody to be 0%, and the value of the well in whichonly Excell 325 medium was added without adding an antibody to be 100%.The results are shown in FIG. 3.

As shown in FIG. 3, the 18C1 antibody did not inhibit at all any signalsby human BMP2, human BMP4, human BMP6, human BMP7, human BMP9, humanBMP15, human GDF5, and human GDF7, and therefore was found to be anantibody that specifically inhibits only a signal by human BMP10.

[Example 5] Isolation of Gene Sequences Encoding VH and VL of Anti-BMP10Monoclonal Antibody

5-1) Preparation of Total RNA from Anti-BMP10 MonoclonalAntibody-Producing Hybridoma Cells

The total RNA of each of the hybridomas was prepared from 1×10⁶ cells ofthe hybridomas that produce the 18C1 antibody, the 12H3 antibody, andthe 11H10 antibody described in Example 1 using Maxwell 16 LEV simplyRNATissue kit (manufactured by Promega Corporation, #AS1280).

5-2) Cloning of Genes of VH and VL of Anti-BMP10 Monoclonal Antibody

From 1 μg of the total RNA of each of the hybridomas obtained in Example5-1, cDNAs were prepared using SMARTer RACE cDNA Amplification Kit(manufactured by Clontech Laboratories, Inc., Cat #634924). By using theobtained cDNA as a template, sequence determination of the cDNA of VHwas carried out by combining Universal Primer A Mix (containing aforward primer) attached to the kit and a reverse primer encoding a ratIgG1 or IgG2a heavy chain constant region.

Specifically, a PCR reaction was carried out using a primer specific torat IgG1 (SEQ ID NO: 1) or a primer specific to rat IgG2a (SEQ ID NO: 2)by combining each with Universal Primer A, whereby a cDNA fragment of VHof each antibody was amplified.

Further, PCR was carried out using a primer specific to rat Ig(κ) (SEQID NO: 3) or a primer specific to rat Ig(λ) (SEQ ID NO: 4) in the samemanner by combining each with Universal Primer A, whereby a cDNAfragment of VL of each antibody was amplified.

In the PCR, a reaction cycle composed of 94° C. for 30 seconds and 72°C. for 3 minutes was carried out 5 times, a reaction cycle composed of94° C. for 30 seconds, 70° C. for 30 seconds, and 72° C. for 3 minuteswas carried out 5 times, and a reaction cycle composed of 94° C. for 30seconds, 68° C. for 30 seconds, and 72° C. for 3 minutes was carried out25 times.

As a result of carrying out agarose gel electrophoresis, in the case ofthe cDNA derived from the 18C1 antibody hybridoma, a PCR amplificationproduct was obtained when a specific primer encoding the IgG1 heavychain constant region was used. In the case of the cDNAs derived fromthe 12H3 antibody-producing and 11H10 antibody hybridomas, PCRamplification products were obtained when a specific primer encoding theIgG2a heavy chain constant region was used.

Further, in the case of the cDNAs derived from the 12H3antibody-producing hybridoma and 11H10 antibody-producing hybridoma, PCRamplification products were obtained also when the primer specific torat Ig(κ) was used. In the case of the cDNA derived from the 18C1antibody-producing hybridoma, a PCR amplification product was obtainedalso when the primer specific to rat Ig(λ) was used. Each of the PCRamplification products was purified using Gel Extraction Kit (QIAEX II,manufactured by QIAGEN, Inc., Cat #20021).

The obtained gene fragment was inserted into a pCR4 vector (manufacturedby Invitrogen, Inc.) using Zero Blunt TOPO PCR Cloning Kit forSequencing (manufactured by Invitrogen, Inc., Cat #K287540SP).

The obtained plasmid was introduced into an E. coli DH5α strain. Theplasmid was extracted from the obtained transformant using an automaticplasmid extractor (manufactured by Kurabo Industries, Ltd.), and thenucleotide sequence was analyzed. As a result, it was confirmed thatfull-length VH cDNA and VL cDNA, in which an ATG sequence presumed to bea start codon is present at the 5′ end of the cDNA, were obtained.

5-3) Analysis of Gene Sequence of Anti-Human BMP10 Monoclonal Antibody VRegion

The entire nucleotide sequences of VHs of the 18C1 antibody, the 12H3antibody, and the 11H10 antibody obtained in Example 5-2 are representedby SEQ ID NOs: 5, 6, and 7, respectively, the entire amino acidsequences of VHs including a signal sequence deduced from the sequencesare represented by SEQ ID NOs: 8, 9, and 10, respectively, the entirenucleotide sequences of VLs thereof are represented by SEQ ID NOs: 11,12, and 13, respectively, and the entire amino acid sequences of VLsincluding a signal sequence deduced from the sequences are representedby SEQ ID NOs: 14, 15, and 16, respectively.

Further, the nucleotide sequences excluding the signal sequence from SEQID NOs: 5, 6, and 7 are represented by SEQ ID NOs: 17, 18, and 19,respectively, the nucleotide sequences excluding the signal sequencefrom SEQ ID NOs: 11, 12, and 13 are represented by SEQ ID NOs: 20, 21,and 22, respectively, the amino acid sequences excluding the signalsequence from SEQ ID NOs: 8, 9, and 10 are represented by SEQ ID NOs:23, 24, and 25, respectively, and the amino acid sequences excluding thesignal sequence from SEQ ID NOs: 14, 15, and 16 are represented by SEQID NOs: 26, 27, and 28, respectively.

By the comparison with the sequence data of known rat antibodies[SEQUENCES of Proteins of Immunological Interest, US Dept. Health andHuman Services (1991)], it could be confirmed that the respectiveisolated cDNAs are full-length cDNAs encoding the 18C1 antibody, the12H3 antibody, and the 11H10 antibody including a secretory signalsequence.

The CDRs of VH and VL of the 18C1 antibody, the 12H3 antibody, and the11H10 antibody were identified by comparison with the amino acidsequences of known antibodies. The amino acid sequences of CDR1, CDR2,and CDR3 of VH of the 18C1 antibody are represented by SEQ ID NOs: 29,30, and 31, respectively, and the amino acid sequences of CDR1, CDR2,and CDR3 of VL thereof are represented by SEQ ID NOs: 32, 33, and 34,respectively. The amino acid sequences of CDR1, CDR2, and CDR3 of VH ofthe 12H3 antibody are represented by SEQ ID NOs: 35, 36, and 37,respectively, and the amino acid sequences of CDR1, CDR2, and CDR3 of VLthereof are represented by SEQ ID NOs: 38, 39, and 40, respectively. Theamino acid sequences of CDR1, CDR2, and CDR3 of VH of the 11H10 antibodyare represented by SEQ ID NOs: 41, 42, and 43, respectively, and theamino acid sequences of CDR1, CDR2, and CDR3 of VL thereof arerepresented by SEQ ID NOs: 44, 45, and 46, respectively.

5-4) Production of Anti-BMP10 Chimeric Antibody

In the recombinant expression of 18C1 chimeric antibody, anN5LG4PE(R409K) vector having a human λ-type light chain constant regionand a human IgG4 modified-type heavy chain constant region was used, andin the recombinant expression of 12H3 and 11H10 chimeric antibodies, anN5KG4PE(R409K) vector having a human κ-type light chain constant regionand a human IgG4 modified-type heavy chain constant region was used. Avector in which a modification of substituting Arg at position 409according to the EU-index of the human IgG4 heavy chain constant regionwith Lys was introduced in the N5KG4PE vector backbone used in WO2006033386 was produced and named N5KG4PE(R409K) vector. In addition, avector in which a human κ chain constant region part was substitutedwith a human λ constant region in this vector was named N5LG4PE(R409K)vector. The cDNAs encoding VH and VL were inserted into each of theabove-mentioned antibody expression vectors. VH was inserted between theSalI and NheI sites, and VL was inserted between the BgIII and BlpI(λ-type) sites or the BgIII and BsiWI (κ-type) sites.

The nucleotide sequence of VH and the nucleotide sequence of VL of the18C1 antibody inserted are represented by SEQ ID NOs: 5 and 11,respectively, and the amino acid sequence of VH and the amino acidsequence of VL expressed thereby are represented by SEQ ID NOs: 8 and14, respectively. The nucleotide sequence of VH and the nucleotidesequence of VL of the 12H3 antibody inserted are represented by SEQ IDNOs: 6 and 12, respectively, and the amino acid sequence of VH and theamino acid sequence of VL expressed thereby are represented by SEQ IDNOs: 9 and 15, respectively. The nucleotide sequence of VH and thenucleotide sequence of VL of the 11H10 antibody inserted are representedby SEQ ID NOs: 7 and 13, respectively, and the amino acid sequence of VHand the amino acid sequence of VL expressed thereby are represented bySEQ ID NOs: 10 and 16, respectively.

PCR was carried out using primers composed of nucleotide sequencesrepresented by SEQ ID NOs: 53 to 56, respectively, in the amplificationof the nucleotide sequences of VL and VH of the 18C1 antibody, primerscomposed of nucleotide sequences represented by SEQ ID NOs: 57 to 60,respectively, in the amplification of the nucleotide sequences of VL andVH of the 12H3 antibody, and primers composed of nucleotide sequencesrepresented by SEQ ID NOs: 61 to 64, respectively, in the amplificationof the nucleotide sequences of VL and VH of the 11H10 antibody.

A recombinant chimeric antibody was expressed using the producedexpression vector and Expi293F Expression System Kit (manufactured byLife Technologies Corporation). The antibody was purified from theculture supernatant using MabSelect SuRe (manufactured by GE Healthcare,Inc.). After replacing the buffer with D-PBS(−) (manufactured by NacalaiTesque, Inc., Cat #14249-24) using a NAP-25 column (manufactured by GEHealthcare, Inc.), the concentration of the antibody was determined bymeasuring the absorbance at 280 nm. As the molecular extinctioncoefficient, 1.50 mL/(mg·cm) was used.

5-5) Evaluation of Binding Activity of Anti-BMP10 Chimeric Antibody toHuman BMP10 Protein by Biacore

For the purpose of comparing the binding activity to human BMP10 betweenthe anti-BMP10 chimeric antibody obtained in Example 5-4 and MAB2926 (R& D Systems, Inc.) that is a known antibody, the binding activity to ahuman BMP10 mature dimer (manufactured by R & D Systems, Cat #2926-BP)was measured by a surface plasmon resonance method (SPR method) usingBiacore T100 (manufactured by GE Healthcare Bio-Sciences Corporation).

The binding activity of the chimeric antibody was measured as follows.An anti-human IgG antibody was immobilized on a CMS sensor chip(manufactured by GE Healthcare Bio-Sciences, Inc., BR100530) using HumanAntibody Capture Kit (manufactured by GE Healthcare Bio-SciencesCorporation, Cat #BR-1008-39) according to the attached protocol. To aflow cell in which the anti-human IgG antibody was immobilized, the 18C1chimeric antibody (hereinafter referred to as ch18C1 antibody) or the12H3 chimeric antibody (hereinafter referred to as ch12H3 antibody) orthe 11H10 chimeric antibody (hereinafter referred to as ch11H10antibody) prepared at 5 μg/mL was added at a flow rate of 10 μL/min for10 seconds.

Further, subsequently, human mature BMP10 prepared at 5 concentrationsby 3-fold serial dilution from 300 ng/mL was added thereto at a flowrate of 30 μL/min, and a binding reaction was monitored for 1 minute anda dissociation reaction was monitored for 30 minutes. The obtainedsensorgram was analyzed using Bia Evaluation Software (manufactured byGE Healthcare Bio-Sciences Corporation), and the kinetic constant ofeach antibody was calculated.

The binding activity of MAB2926 was measured as follows. An anti-mouseIgG antibody was immobilized on a CMS sensor chip (manufactured by GEHealthcare Bio-Sciences, Inc., BR100530) using Mouse Antibody CaptureKit (manufactured by GE Healthcare Bio-Sciences Corporation, Cat#BR-1008-38) according to the attached protocol. To a flow cell in whichan anti-mouse IgG antibody was immobilized, MAB2926 prepared at 5 μg/mLwas added at a flow rate of 10 μL/min for 10 seconds.

Further, subsequently, human mature BMP10 protein prepared at 5concentrations by 3-fold serial dilution from 300 ng/mL was addedthereto at a flow rate of 30 μL/min, and a binding reaction wasmonitored for 1 minute and a dissociation reaction was monitored for 30minutes. The obtained sensorgram was analyzed using Bia EvaluationSoftware (manufactured by GE Healthcare Bio-Sciences Corporation), andthe kinetic constant of MAB2926 was calculated. The calculatedassociation rate constant (ka), dissociation rate constant (kd), anddissociation constant [kd/ka=K_(D)] of each antibody are shown in Table1.

TABLE 1 Antibody name ka kd KD MAB2926 4.06E+07 0.001343 3.31E−11ch11H10 antibody 3.22E+07 2.02E−04 6.26E−12 ch12H3 antibody 1.31E+071.54E−04 1.18E−11 ch18C1 antibody 3.88E+07 1.02E−04 2.62E−12 * kaexceeds the detection limit of the apparatus, and therefore, the valueis shown for reference only.

As shown in Table 1, it was revealed that the obtained ch18C1 antibody,ch12H3 antibody, and ch11H10 antibody exhibit stronger binding than theknown antibody MAB2926.

[Example 6] Analysis of Receptor with which Obtained Antibody Competes6-1) Preparation of ALK1-Fc

ALK1-Fc was prepared according to the method described in Example 1 ofWO 2010/126169.

6-2) Analysis of Competing Receptor by Biacore

BMP10 transmits a signal by binding to two receptors: type I and typeII. In order to analyze which receptor the obtained anti-BMP10 antibodycompetes with, measurement was carried out by a surface plasmonresonance method (SPR method) using Biacore T100 (manufactured by GEHealthcare Bio-Sciences Corporation).

An anti-human IgG antibody was immobilized on a CMS sensor chip(manufactured by GE Healthcare Bio-Sciences, Inc., BR100530) using HumanAntibody Capture Kit (manufactured by GE Healthcare Bio-SciencesCorporation, Cat #BR-1008-39) according to the attached protocol.

To a flow cell in which the anti-human IgG antibody was immobilized, anyof ALK1-Fc, BMPR2-Fc (manufactured by R & D Systems, Inc., Cat#811-BR-100), and Endoglin-Fc (manufactured by R & D Systems, Inc., Cat.#6578-EN-025) prepared at 10 μg/mL was added at a flow rate of 10 μL/minfor 10 seconds.

Further, subsequently, a mixture of human mature BMP10 (manufactured byR & D Systems, Inc., Cat #2926-BP) prepared at 100 ng/mL and 1 μg/mL ofan anti-BMP10 antibody was added thereto at a flow rate of 10 μL/min for30 seconds.

The results are shown in Table 2. In Table 2, a case where the mixtureof the antibody with the mature BMP10 binds to the captured ALK1-Fc,BMPR2-Fc, or Endoglin-Fc is denoted by “+”, and a case where it does notbind thereto is denoted by “−”.

TABLE 2 Antibody name ALK1-Fc BMPR2-Fc Endoglin-Fc MAB2926 − + + 11H10antibody + − − 12H3 antibody + − − 18C1 antibody + − −

As shown in Table 2, it can be said that in the case where the mixtureof the mature BMP10 and the antibody does not bind to the capturedreceptor, the antibody and the receptor compete with each other. As aresult, it was found that MAB2926 inhibits the binding of BMP10 toALK1-Fc.

On the other hand, MAB2926 did not inhibit the binding of BMP10 toBMPR2-Fc and Endoglin-Fc. The 18C1 antibody, the 12H3 antibody, and the11H10 antibody all did not inhibit the binding of BMP10 to ALK1-Fc. Onthe other hand, the 18C1 antibody, the 12H3 antibody, and the 11H10antibody all inhibited the binding of BMP10 to BMPR2-Fc and Endoglin-Fc.

From the above results, it was revealed that the 18C1 antibody, the 12H3antibody, and the 11H10 antibody are antibodies having a novelinhibitory mode of inhibiting a different receptor from the knownantibody.

[Example 7] Evaluation of Effect of Obtained Antibody on Blood Pressureof Normal Animal

The effect of the 18C1 antibody on blood pressure was evaluated using SDrats. Male SD rats (manufactured by Charles River Laboratories Japan,Inc.) at 6 weeks of age were purchased and used for an experiment. Therats were given sterile tap water as drinking water and chow FR-2(manufactured by Funabashi Farm Co., Ltd.) as feed ad libitum. Afteracclimation, the rats underwent surgery for implantation of a telemetrytransmitter at 7 weeks of age.

Specifically, the rats were anesthetized by administering 50 mg/kg ofpentobarbital sodium (manufactured by Tokyo Chemical Industry Co., Ltd.)to the abdominal cavity of each rat. A blood pressure sensor of atelemetry transmitter (TA11PA-C40, Data Sciences International) wasinserted into the abdominal aorta, and the transmitter body wasimplanted into the abdominal cavity. By observation of generalconditions and hemodynamic monitoring, it was confirmed that the ratswere recovered satisfactorily from the effect of the surgery. Theanimals implanted with the telemetry transmitter were grouped using theaverage value of the systolic blood pressure as an index. Administrationof an antibody was carried out at 12 weeks of age.

Specifically, a solution in which the 18C1 antibody was prepared at 1mg/mL with PBS was administered at a dose of 1 mL/kg. Further, asolution in which the 12H3 antibody or the 11H10 antibody was preparedat 5 mg/mL with PBS was administered at a dose of 1 mL/kg. Theadministration was carried out through a subcutaneous route. The signalof the blood pressure waveform sent from the transmitter was received bya receiving board (RPC-1, Data Sciences International) installed underthe cage. The signal was incorporated into a data acquisition/analysissystem (DATAQUEST ART Gold ver. 2.30, Data Sciences International) viaData Exchange Matrix (Data Sciences International), and the measurementdata of the systolic blood pressure for 10 seconds was obtained every 5minutes. The results are shown in FIG. 4A to FIG. 4C.

As shown in FIG. 4A and FIG. 4B, a decrease in the systolic bloodpressure was observed by the administration of the 18C1 antibody or the12H3 antibody. Further, the antihypertensive effect of the 18C1 antibodywas stronger than that of the 12H3 antibody. On the other hand, as shownin FIG. 4C, in the administration of the 11H10 antibody, an effect onthe systolic blood pressure was not observed. From the above results, itwas newly found that the anti-BMP10 antibody has an antihypertensiveeffect.

In addition, it was found that the antihypertensive effect correlateswith the BMP10 neutralizing activity in vitro of the antibody, and inthe 11H10 antibody having a relatively weak neutralizing activity, anantihypertensive effect is not observed. Based on this, it was newlyfound that only an antibody having a strong BMP10 neutralizing activityhas an antihypertensive effect.

[Example 8] Evaluation of Effect of 18C1 Antibody on Blood Pressure ofSpontaneously Hypertensive Rat

In order to examine the antihypertensive effect of the obtained antibodyon a hypertensive animal, the effect of the 18C1 antibody on bloodpressure was evaluated using male spontaneously hypertensive rats(SHR/Izm, Japan SLC, Inc.).

Male SHR/Izm rats (Japan SLC, Inc.) at 14 weeks of age were purchasedand used for an experiment. The rats were given sterile tap water asdrinking water and chow FR-2 (manufactured by Funabashi Farm Co., Ltd.)as feed ad libitum. After acclimation, the rats underwent surgery forimplantation of a telemetry transmitter at 16 weeks of age. The methodfor the surgery for implantation of a telemetry transmitter and theacquisition of the blood pressure waveform were carried out according toExample 7. A solution in which the 18C1 antibody was prepared at 5 mg/mLwith PBS was administered to the rats at 21 weeks of age at a dose of 1mL/kg. The administration was carried out through a subcutaneous route.The results are shown in FIG. 5.

As shown in FIG. 5, by the administration of the 18C1 antibody at 5mg/kg, it was observed that the systolic blood pressure is persistentlydecreased during about one month. From the above results, it wasdemonstrated that neutralization of BMP10 has a therapeutic effect onhypertensive pathology.

[Example 9] Evaluation of Effect of 18C1 Antibody on Dahl Salt-SensitiveRat

The effect of the BMP10 antibody on hypertension and a cardiac or renaldisorder induced by giving high salt to Dahl salt-sensitive rats(hereinafter, Dahl-S rats) was evaluated. Male Dahl salt-sensitive rats(DIS/Eis:Slc) (manufactured by Japan SLC, Inc.) at 5 weeks of age werepurchased and used for an experiment.

The rats were given FR-2 feed (manufactured by Funabashi Farm Co., Ltd.)until 6 weeks of age, and thereafter given a high salt feed (8%NaCl-containing FR-2 feed, manufactured by Oriental Yeast Co., Ltd.). Asa normal control group, Dahl-S rats given a normal diet (FR-2 feed) alsoafter 6 weeks of age were used. As drinking water, drinking water foranimals was given ad libitum.

The blood pressure and heart rate were measured at 8 weeks of age. Themeasurement of the blood pressure and heart rate was carried out aftereach acclimation for measurement twice using a mouse-rat non-invasivesphygmomanometer (BP-98E, manufactured by Softron Co., Ltd.). The highsalt feed group was divided into two groups each consisting of 12 ratsusing the body weight, blood pressure, and heart rate at 8 weeks of ageas indices, and the vehicle (PBS 1 mL/kg) or the 18C1 antibody at a doseof 5 mg/kg was subcutaneously administered at a frequency of once aweek.

At 16 weeks of age, measurement of the blood pressure, echocardiography,and measurement of urine parameters were carried out. Further, the bloodwas collected from the abdominal aorta under isoflurane inhalationanesthesia, and autopsy was carried out after the rats were sacrificedby bleeding. The heart, lung, and bilateral kidneys were excised, andthe tissue weight was measured.

The echocardiography was carried out using an ultrasound high-resolutionimaging system for small animals (Vevo 2100, manufactured byVisualSonics, Inc.) and a high frequency high frame rate probe (MS-200,manufactured by VisualSonics, Inc.) with ultrasonic waves at a centralfrequency of 15.0 MHz after shaving the anterior thorax of each ratunder isoflurane inhalation anesthesia. The left ventricular posteriorwall thickness (LVPW; s, LVPW; d) was measured at the papillary musclelevel by the M-mode method. The mitral annulus velocity (e′) wasmeasured by a tissue Doppler method in which a sample volume was placedon the mitral annulus.

Urine collection was carried out for 24 hours in a metabolic cage(T-480, manufactured by Tokiwa Kagaku Kikai Co., Ltd.) with feeding anddrinking ad libitum. A urine sample was centrifuged at 1870×g and 4° C.for 15 minutes after the urine volume was measured, and the supernatantwas used for measurement. The urine parameters were measured using anautomatic analyzer Hitachi 7170S (manufactured by Hitachi, Ltd.) or afully automatic electrolyte analyzer [PVA-EXII manufactured by A & TCo., Ltd.].

The collected heart, aorta, and kidney were subjected to ahistopathological analysis. Specifically, the collected heart, aorta,and kidney were fixed with a 10 vol % neutral buffered formalinsolution. A section was prepared from a paraffin-embedded blockaccording to a usual method and subjected to Masson trichrome (MT)staining or Hematoxylin-Eosin (HE) staining.

As for the renal glomerular damage pathology score, HE-stained specimenswere observed under a light microscope, and the area ratio of lesion perglomerulus (mesangial expansion, glomerular sclerosis, and/or glomerularcapillary collapse) with respect to 100 or more glomeruli as testsubjects for each individual was graded into the following five levels(0: normal, 1: 1 to 25%, 2: 26 to 50%, 3: 51 to 75%, and 4: 76 to 100%).Thereafter, a glomerular lesion score (the sum of “the score points ofeach grade” x “the ratio of glomeruli for each grade”) for eachindividual was calculated. In a statistical analysis, the number ofglomeruli with a score of 1 or more was compared.

As for a renal tubulointerstitial damage pathology score, HE-stainedspecimens were observed under a light microscope, and the ratio of renaltubular and interstitial lesions (basophilic tubule, hyaline cast,interstitial inflammation and/or tubular dilatation) in each section tothe area of each section was graded into the following five levels (0:normal, 1: 1 to 25%, 2: 26 to 50%, 3: 51 to 75%, and 4: 76 to 100) foreach individual.

The results are shown in FIGS. 6 to 13.

As shown in FIG. 6, the blood pressure of the Dahl-S rats was remarkablyincreased by the high salt diet. By administration of the 18C1 antibody,the high blood pressure of the Dahl-S rats induced by the high salt dietwas reduced to a level equivalent to that of the normal diet group.

Further, as shown in FIG. 7A and FIG. 7B, both the blood sodium leveland the urine sodium excretion of the Dahl-S rats were increased by thehigh salt diet. By administration of the 18C1 antibody, the urine sodiumexcretion was further increased. In addition, by administration of the18C1 antibody, the blood sodium level was normalized to a levelequivalent to that of the normal diet group. From the above results, itwas revealed that the anti-BMP10 antibody exhibits a remarkable effecton sodium retention and high blood pressure caused by the high salt dietin the Dahl-S rats.

On the other hand, as shown in FIG. 8, the urine volume of the Dahl-Srats was increased by the high salt diet and decreased by the anti-BMP10antibody. That is, it was demonstrated that the anti-BMP10 antibody hasan effect of normalizing the renal sodium excretion disorder in thesalt-sensitive pathological conditions, and it was suggested that theantibody has a mode of action different from a diuretic agent whichincreases the urine volume.

As shown in FIG. 9, an increase in urine protein excretion was observedin the high salt diet group as compared with the normal diet group. Inaddition, as shown in FIG. 10A, FIG. 10B, and FIG. 11B, hyaline casts,basophilic tubules, and tubular dilation were observed in the kidney inthe high salt diet group, and it was confirmed that the renaltubulointerstitium is remarkably impaired. Further, as shown in FIG.10D, FIG. 10E, and FIG. 11A, a glomerular hyaline deposit was observedin the high salt diet group, and it was confirmed that the renalglomerulus was remarkably impaired. On the other hand, as shown in FIG.9, FIG. 10C, and FIG. 10F, in the high salt diet+18C1 antibodyadministration group, the increase in urine protein excretion, the renalglomerular disorder, and the renal tubulointerstitial disorder weredramatically suppressed.

From the above results, it was suggested that the anti-BMP10 antibodyhas a therapeutic effect on a glomerular disorder and atubulointerstitial disorder caused by high blood pressure.

As shown in FIG. 12A, thickening of the left ventricular posterior wallwas observed in the high salt diet group as compared with the normaldiet group. Further, as shown in FIG. 12B, in the high salt diet group,a decrease in the index e′ of the left ventricular diastolic functionwas observed as compared with the normal diet group, and it wasdemonstrated that cardiac diastolic dysfunction has occurred.

As shown in FIG. 13, an increase in the lung weight was observed in thehigh salt diet group, and it was suggested that pulmonary congestion dueto left ventricular diastolic dysfunction has occurred. On the otherhand, as shown in FIG. 12A, FIG. 12B, and FIG. 13, in the high saltdiet+18C1 antibody administration group, the left ventricular posteriorwall thickness, e′, and lung weight were maintained at a levelequivalent to that of the normal diet group.

From the above results, it was suggested that the anti-BMP10 antibodyhas a therapeutic effect on diastolic heart failure caused by high bloodpressure.

[Example 10] Evaluation of Neutralizing Activity of Anti-BMP10 AntibodyAgainst Human Serum

As a ligand for ALK1, BMP9 is also known other than BMP10. For thepurpose of evaluating the neutralizing activity against BMP9 and BMP10in blood, the neutralizing activity against human ALK1 expressingreporter cells when stimulating with human serum was compared.

10-1) Acquisition of 10D5 Antibody that is Anti-BMP9 Antibody

An anti-BMP9 antibody 10D5 was prepared according to the methoddescribed in Example 7 of WO 2014/007198.

10-2) Comparison of BMP10 Neutralizing Activity Between ObtainedAntibody and Known Antibody

To a 96-well fluorescence and luminescence plate (manufactured byCorning, Inc., Cat #3916), human serum (Human True A serum, pool ofdonors, manufactured by Biopredic International, Cat #SER019) was addedat a final concentration of 5%.

Subsequently, the 18C1 antibody, the 12H3 antibody, or the 11H10antibody, each of which is an anti-BMP10 antibody, the 10D5 antibody,which is an anti-BMP9 antibody, or a control antibody (Purified Rat IgG1Isotype control, manufactured by BD, Inc., Cat #553993), or a mixture ofan anti-BMP9 antibody and an anti-BMP10 antibody was added thereto. Asthe mixture of an anti-BMP9 antibody and an anti-BMP10 antibody, amixture of the 12H3 antibody and the 10D5 antibody or a mixture of the11H10 antibody and the 10D5 antibody was used.

As for the antibody, each antibody was prepared at 5 serialconcentrations by 3-fold dilution from the final concentration of 10000ng/mL regardless of whether it is mixed or not, and added. Thereafter,an ALK1/Id1-Luc/CHO cell suspension was added at 5×10⁴ cells/well. Allsamples were diluted with Excell 325 medium so as to achieve 100 μL/wellin the end when combining the human serum, the antibody dilutionsolution, and the cell suspension. The liquid in the well was madeuniform with a plate mixer, and the cells were cultured at 37° C. for 20hours.

After 20 hours, an assay solution of Nano-Glo Luciferase Assay preparedaccording to the package insert was added at 40 μL/well, followed bystirring, and then, a luciferase activity was measured using Glomax(manufactured by Promega Corporation). The neutralizing activity (%) ofthe antibody was calculated by setting the value of the well in whichonly the serum was added without adding an antibody to be 0%, and thevalue of the well in which only Excell 325 medium was added withoutadding an antibody to be 100%.

As a result, as shown in FIG. 14, the control antibody, the 12H3antibody and the 11H10 antibody, each of which is an anti-BMP10antibody, and the 10D5 antibody, which is an anti-BMP9 antibody all didnot exhibit a neutralizing activity against human serum. On the otherhand, the mixture of the 12H3 antibody and the 10D5 antibody and themixture of the 11H10 antibody and the 10D5 antibody exhibited a distinctneutralizing activity against human serum.

From the above results, it was suggested that a human BMP9/BMP10heterodimer is present in human serum. Further, the 18C1 antibody whichis an anti-BMP10 antibody exhibited a neutralizing activity againsthuman serum alone. This result suggested that the 18C1 antibody is anovel antibody having a strong neutralizing activity against a humanBMP9/BMP10 heterodimer present in human serum.

[Example 11] Detection of BMP9/BMP10 Heterodimer in Human Blood

In order to detect the BMP9/BMP10 heterodimer suggested in Example 10,sandwich ELISA was carried out using an anti-BMP9 antibody and ananti-BMP10 antibody.

11-1) Biotinylation of 12H3 Antibody

The 12H3 antibody which is an anti-BMP10 antibody was biotinylated forbeing used as a detection antibody in sandwich ELISA. The biotinylationwas carried out using Biotin Labeling Kit-NH2 kit (Cat #LK03)manufactured by Dojindo Laboratories. The method was carried out inaccordance with the attached document described on the product.

11-2) Sandwich ELISA Using Anti-BMP9 Antibody and Anti-BMP10 Antibody

A solution in which the 10D5 antibody which is an anti-BMP9 antibody,the 11H10 antibody which is an anti-BMP10 antibody, or a controlantibody (Purified Mouse IgG1 κ Isotype control, manufactured by BD,Inc., Cat #554121) was diluted to 3 μg/mL with a carbonate-bicarbonatebuffer (50 mM NaHCO₃ pH 9.6, Sigma-Aldrich Co. LLC, Cat #C3041) wasadded as an immobilizing solution to a 96-well ELISA plate (F96 MAXISORPNUNC-IMMNO PLATE, manufactured by Thermo Fisher Scientific, Inc., Cat#442404) at 100 μL/well, and adsorption was carried out by leaving theplate to stand overnight at 4° C.

After the antibody solution was removed, 1% BSA-PBS was added at 300μL/well, and blocking was carried out by leaving the plate to stand atroom temperature for 1 hour, followed by washing 5 times with PBST.Subsequently, a solution obtained by diluting human serum (Human True Aserum, pool of donors, manufactured by Biopredic International, Cat#SER019) to a final concentration of 1%, 2%, 4%, 8%, or 16% with 0.1%BSA-PBST was added at 100 μL/well, and the plate was left to stand atroom temperature for 1 hour to cause a reaction, followed by washing 5times with PBST.

Subsequently, a solution in which the biotinylated 12H3 antibodyproduced in Example 11, 11-1) was prepared at 50 ng/mL with 0.1%BSA-PBST was dispensed at 100 μL/well, and the plate was left to standat room temperature for 1 hour. After this plate was washed 5 times withPBST, Streptavidin-PolyHRP80, Pre-diluted in Stabilizer (1/20)(manufactured by Stereospecific Detection Technologies GmbH, Cat#SP80D50) diluted 500 times with 0.1% BSA-PBST was added at 100 μL/well,and the plate was left to stand at room temperature for 1 hour.

The plate was washed 5 times with PBST, a TMB substrate solution (TMB+Substrate-Chromogen, manufactured by Dako, Inc., Cat #S1599) was addedat 50 μL/well so as to develop a color. When appropriate colordevelopment was obtained, a 1 N sulfuric acid solution (manufactured byWako, Cat #192-04755) was added at 50 μL/well, and an absorbance at 450and 570 nm was measured using Multiskan Spectrum (manufactured by ThermoLabsystems, Inc.). The results are sown in FIG. 15.

As shown in FIG. 15, when the control antibody and the 11H10 antibodywhich is an anti-BMP10 antibody were immobilized, a reaction for theserum was not observed. On the other hand, when the 10D5 antibody whichis an anti-BMP9 antibody was immobilized, a distinct reaction wasobserved in a serum concentration dependent manner. From the aboveresults, it was suggested that a BMP9/BMP10 heterodimer that issimultaneously recognized by the anti-BMP9 antibody and the anti-BMP10antibody is present in human serum.

[Example 12] Production of Humanized Antibody of 18C1 Antibody (1)Designing of Amino Acid Sequences of VH and VL of 18C1 HumanizedAntibody

By the method described below, the amino acid sequences of various VHand VL of 18C1 humanized antibodies were designed. In the followingdescription, the 18C1 humanized antibodies having amino acid sequencesof various VH and VL are collectively referred to as hz18C1 antibody. Asthe amino acid sequence of a framework (hereinafter referred to as FR)of a known human antibody suitable for grafting the amino acid sequenceof a CDR of the 18C1 antibody, hSGHII and V1-22 were selected from humanFR consensus sequences and human antibody germline sequences reported byKabat et al. [Sequences of Proteins of Immunological Interest, US Dept.Health and Human Services (1991)], and the CDR was to be grafted into FRthereof.

The amino acid sequences of CDRs 1 to 3 of 18C1 VH represented by SEQ IDNOs: 29, 30, and 31, respectively, were grafted into appropriatepositions of the amino acid sequence of FR of hSGHII, whereby hz18C1 HV0(SEQ ID NO: 70) was designed. In addition, the amino acid sequences ofCDRs 1 to 3 of 18C1 VL represented by SEQ ID NOs: 32, 33, and 34,respectively, were grafted into appropriate positions of the amino acidsequence of FR of V1-22 (as FR4, the FR4 of the 18C1 chimeric antibodywas used as it is), whereby hz18C1 LV0 (SEQ ID NO: 71) was designed.

By computer modeling of hz18C1 HV0 and hz18C1 LV0 designed as describedabove, amino acid residues of FR considered to affect the bindingactivity of the antibody were identified. As a result, among the aminoacid residues of FR of the variable region of the hz18C1 LV0HV0antibody, as the amino acid residues considered to change thethree-dimensional structure of an antigen-binding site so as to affectthe binding activity of the antibody, in VH, Pro at position 14, Leu atposition 20, Gly at position 27, Val at position 29, Ser at position 30,Ile at position 37, Ile at position 48, Val at position 67, Val atposition 71, Asn at position 76, Phe at position 78, Leu at position 82,Val at position 85, Val at position 92, Tyr at position 94, and Thr atposition 109 of the amino acid sequence represented by SEQ ID NO: 70,and in VL, Pro at position 7, Val at position 10, Glu at position 12,Pro at position 14, Lys at position 16, Thr at position 19, Ile atposition 20, Pro at position 41, Val at position 48, Ser at position 75,Leu at position 81, Lys at position 82, Asp at position 88, and Tyr atposition 90 of the amino acid sequence represented by SEQ ID NO: 71 wereselected, respectively. Among the selected amino acid residues, at leastone or more amino acid residues were substituted with amino acidresidues present at the same site of the 18C1 antibody, therebydesigning VH and VL of humanized antibodies having various alterations.

In addition to the designing of standard CDR grafting as describedabove, in some VH or VL, an alteration of VH CDR was also carried out.In the VH of the hz18C1 antibody in which the alteration of VH CDR wascarried out, at least one alteration of an alteration of substitutingVal at position 4 in the amino acid sequence of CDR1 of VH representedby SEQ ID NO: 29 is substituted with Ala, and an alteration ofsubstituting Ser at position 16 in CDR2 of VH represented by SEQ ID NO:30 with Asp was introduced.

Specific amino acid sequences as the amino acid sequence designedaccording to the above process are shown in FIG. 16 for VH and in FIG.18A for VL.

Further, other than the above-mentioned CDR grafting method, by asurface reconstruction method (Proc. Natl. Acad. Sci. USA, 1994, 91(3):969-73, and Protein Engineering, 1996, 10, 895-90), VL and VH ofhumanized antibodies having various alterations were designed bysubstituting an amino acid residue in FR considered not to affect thebinding activity of an antibody with an amino acid residue considered tolower the antigenicity from a model structure of the variable region ofthe 18C1 antibody constructed above. In some VL or VH, an alteration ofVH CDR as mentioned with respect to the case of the CDR grafting methodwas also carried out.

Specifically, at least one alteration of amino acid alterations ofsubstituting Val at position 3 with Ala, Asn at position 8 with Asp, Leuat position 14 with Ala, Lys at position 19 with Thr, Phe at position 75with Ser, Asn at position 80 with Asp, Ile at position 83 with Val, andIle at position 88 with Val was introduced in the amino acid sequencerepresented by SEQ ID NO: 26. By doing this, hz18C1 VLres01 to 16composed of the amino acid sequences represented by SEQ ID NOS: 72 to87, respectively, were designed as the VL of the hz18C1 antibody and areshown in FIG. 18B.

Further, with respect to VH, at least one alteration of amino acidalterations of substituting Leu at position 11 with Ala, Leu at position14 with Pro, Ser at position 19 with Asp, Phe at position 27 with Ala,Val at position 34 with Ala, Ser at position 65 with Asp, Ser atposition 68 with Ala, Arg at position 71 with Lys, Gln at position 77with Glu, Phe at position 79 with Ala, Asn at position 83 with Asp, Leuat position 85 with Asp, and His at position 107 with Gln was introducedin the amino acid sequence represented by SEQ ID NO: 23. As the VH ofthe hz18C1 antibody designed in this manner, hz18C1 VHres01 to 32 areshown in FIG. 17. With respect to hz18C1 VHres16, the amino acidsequence thereof is represented by SEQ ID NO: 98.

In addition, as the amino acid sequence of VH of a humanized antibody,an amino acid sequence of VH was designed using a CDR grafting methodand a surface reconstruction method in combination. Specifically, atleast one alteration of amino acid alterations of substituting Gly atposition 10 with Asp, Lys at position 13 with Gln, Ser at position 19with Asp, Leu at position 20 with Ile, Gly at position 27 with Phe, Valat position 29 with Leu, Ser at position 30 with Thr, Ile at position 37with Val, Ile at position 48 with Met, Ser at position 65 with Asp, Valat position 67 with Leu, Thr at position 68 with Ala, Val at position 71with Arg, Asn at position 76 with Ser, Gln at position 77 with Glu, Pheat position 78 with Val, Ser at position 79 with Phe, Leu at position 82with Met, Ser at position 83 with Asp, Val at position 85 with Leu, Thrat position 86 with Gln, Ala at position 87 with Thr, Ala at position 88with Asp, Val at position 92 with Lys, Tyr at position 94 with Phe, Thrat position 109 with Ile, and Leu at position 110 with Met wasintroduced in the amino acid sequence represented by SEQ ID NO: 70. Bydoing this, hz18C1 HVmut01 to 10 (SEQ ID NOs: 88 to 97) were designed asthe VH of the hz18C1 antibody, and the amino acid sequences thereof areshown in FIG. 19. Among these, CDRs 1 to 3 of the hz18C1 HVmut01 to 04all contain the same amino acid sequences (amino acid sequencesrepresented by SEQ ID NOs: 29 to 31) as those of the 18C1 antibody. Onthe other hand, the hz18C1 HVmut05 to 10 contain a mutation in CDR2.That is, CDRs 1 and 3 of the hz18C1 HVmut05 to 10 contain amino acidsequences represented by SEQ ID NOs: 29 and 31, respectively, and CDR2thereof contains an amino acid sequence (SEQ ID NO: 99) in which Ser atposition 16 of the amino acid sequence represented by SEQ ID NO: 30 issubstituted with Asp.

(2) Designing of Gene of Variable Region of Humanized Antibody

Nucleotide sequences encoding amino acid sequences of the variableregion of humanized antibodies shown in Table 3 were designed using acodon used highly frequently in animal cells.

TABLE 3 Antibody name VH VL SEQ ID NO of VH SEQ ID NO of VLVLres02HVmut07 HVmut07 VLres02 SEQ ID NO: 94 SEQ ID NO: 73VLres02HVmut08 HVmut08 VLres02 SEQ ID NO: 95 SEQ ID NO: 73VLres04HVmut04 HVmut04 VLres04 SEQ ID NO: 91 SEQ ID NO: 75VLres04VHres16 VHres16 VLres04 SEQ ID NO: 98 SEQ ID NO: 75VLres06HVmut02 HVmut02 VLres06 SEQ ID NO: 89 SEQ ID NO: 77VLres06HVmut10 HVmut10 VLres06 SEQ ID NO: 97 SEQ ID NO: 77VLres07HVmut10 HVmut10 VLres07 SEQ ID NO. 97 SEQ ID NO: 78VLres07VHres16 VHres16 VLres07 SEQ ID NO: 98 SEQ ID NO: 78VLres08HVmut04 HVmut04 VLres08 SEQ ID NO: 91 SEQ ID NO: 79VLres08HVmut08 HVmut08 VLres08 SEQ ID NO: 95 SEQ ID NO: 79VLres08VHres16 VHres16 VLres08 SEQ ID NO: 98 SEQ ID NO: 79VLres10HVmut02 HVmut02 VLres10 SEQ ID NO: 89 SEQ ID NO: 81VLres10HVmut04 HVmut04 VLres10 SEQ ID NO: 91 SEQ ID NO: 81VLres10HVmut08 HVmut08 VLres10 SEQ ID NO: 95 SEQ ID NO: 81VLres10HVmut10 HVmut10 VLres10 SEQ ID NO: 97 SEQ ID NO: 81VLres10VHres16 VHres16 VLres10 SEQ ID NO: 98 SEQ ID NO: 81VLres14HVmut07 HVmut07 VLres14 SEQ ID NO: 94 SEQ ID NO: 85VLres14HVmut08 HVmut08 VLres14 SEQ ID NO: 95 SEQ ID NO: 85VLres14HVmut10 HVmut10 VLres14 SEQ ID NO: 97 SEQ ID NO: 85VLres14VHres16 VHres16 VLres14 SEQ ID NO: 98 SEQ ID NO: 85VLres16HVmut07 HVmut07 VLres16 SEQ ID NO: 94 SEQ ID NO: 87VLres16HVmut10 HVmut10 VLres16 SEQ ID NO: 97 SEQ ID NO: 87VLres16VHres16 VHres16 VLres16 SEQ ID NO: 98 SEQ ID NO: 87

(3) Production of Humanized Antibody

A necessary plasmid was produced by introducing a gene fragmentcorresponding to the nucleotide sequence designed in (2) into anexpression vector using a seamless cloning method. However, as a VLexpression vector, a pCI-OtCMV_hL vector having a signal sequence and ahuman λ chain constant region sequence was used, and as a VH expressionvector, a pCI-OtCAG_hG4PE(R409K) vector having a signal sequence and ahuman γ chain constant region sequence was used. The constant regionsequence included in the pCI-OtCAG_hG4PE(R409K) vector is a heavy chainconstant region of an IgG4 mutant obtained by substituting a Ser residueat position 228 according to the EU-index in the heavy chain constantregion of human IgG4 with Pro, a Leu residue at position 235 thereinwith Glu, and an Arg residue at position 409 therein with Lys(hereinafter denoted by IgG4PE R409K (WO 2006/033386)). Note that thesevectors are vectors produced through total synthesis by introducingrestriction enzyme sites necessary for expressing a human antibody geneusing a pCI vector of Promega Corporation as a common main backbone. Thecompleted plasmid was prepared in a large amount using NucleoBond XtraMidi EF Kit (Takara Bio, Inc.). Subsequently, a target humanizedantibody was transiently expressed using Expi293 Expression System Kit(Life Technologies, Inc.). The method for introducing the plasmid wascarried out according to the package insert.

The light chain expression vector and the heavy chain expression vectorwere mixed at a ratio of 1:2 and introduced. The cells after theintroduction of the plasmid were cultured for 3 days under conditions of37° C., 5% CO₂, and 125 rpm. Thereafter, the cell culture suspension wascentrifuged, and the culture supernatant was collected through a 0.2 μmfilter (Thermo Scientific, Inc.). A purified antibody was obtained fromthe culture supernatant by affinity purification using MabSelect SuRe(GE Healthcare, Inc.).

Specifically, after a resin packed in the column was equilibrated withPBS, the culture supernatant was added to the column, washed twice withPBS, and then washed once with Wash buffer 1 (PBS with 1 M NaCl) andonce with Wash buffer 2 (20 mM citric acid and 50 mM NaCl, pH 5.0), andthereafter, the antibody was eluted using an elution buffer (20 mMcitric acid and 50 mM NaCl, pH 3.4). The obtained antibody solution wasneutralized by adding a 1/10 amount of a neutralization buffer (1 Mphosphoric acid-NaOH, pH 7.0), and the solvent of the antibody solutionwas replaced with PBS using NAP 25 (GE Healthcare, Inc.). The antibodysolution after buffer replacement was concentrated by ultrafiltrationusing Amicon Ultra-4 Centrifugal Filter Units (Millipore, Inc.), and anabsorbance A₂₈₀ was measured using Nanodrop (Thermo Scientific, Inc.),and the concentration of the antibody solution was measured andadjusted. The extinction coefficient was calculated based on the aminoacid sequence of each humanized antibody according to the method of C.N. Pace et al. (1995, Prot. Sci. 4: 2411-2423). The purified antibodywas confirmed for its quality by analytical gel filtrationchromatography (using an apparatus manufactured by Shimadzu Corporation,and a column TSKgel SuperSW3000 manufactured by Tosoh Corporation) andSDS-PAGE.

[Example 13] Evaluation of Binding Activity of Anti-BMP10 HumanizedAntibody

For the purpose of comparing the binding activity to human BMP10 betweenthe ch18C1 antibody obtained in Example 5-3 and the anti-BMP10 humanizedantibody obtained in Example 12, the binding activity to a human BMP10mature dimer (manufactured by R & D Systems, Cat #2926-BP) was measuredby a surface plasmon resonance method (SPR method) using Biacore T100(manufactured by GE Healthcare Bio-Sciences Corporation).

The binding activity of the anti-BMP10 antibody was measured as follows.An anti-human IgG antibody was immobilized on a CMS sensor chip(manufactured by GE Healthcare Bio-Sciences, Inc., BR100530) using HumanAntibody Capture Kit (manufactured by GE Healthcare Bio-SciencesCorporation, Cat #BR-1008-39) according to the attached protocol. To aflow cell in which the anti-human IgG antibody was immobilized, theantibody prepared at 5 μg/mL was added at a flow rate of 10 μL/min for10 seconds.

Subsequently, human mature BMP10 prepared at 5 concentrations by 3-foldserial dilution from the 100 ng/mL was added thereto at a flow rate of30 μL/min, and a binding reaction was monitored for 1 minute and adissociation reaction was monitored for 10 minutes. The obtainedsensorgram was analyzed using Bia Evaluation Software (manufactured byGE Healthcare Bio-Sciences Corporation), and the kinetic constant ofeach antibody was calculated. The calculated association rate constant(ka), dissociation rate constant (kd), and dissociation constant[kd/ka=K_(D)] of each antibody are shown in Table 4.

TABLE 4 Antibody name ka kd KD VLres02HVmut07 4.48E+07 1.33E−04 2.97E−12VLres02HVmut08 3.68E+07 1.13E−04 3.05E−12 VLres04HVmut04 4.09E+071.30E−04 3.19E−12 VLres04VHres16 4.45E+07 1.43E−04 3.21E−12VLres06HVmut02 4.06E+07 1.37E−04 3.37E−12 VLres06HVmut10 4.30E+071.26E−04 2.94E−12 VLres07HVmut10 4.51E+07 1.21E−04 2.69E−12VLres07VHres16 4.59E+07 1.43E−04 3.11E−12 VLres08HVmut04 4.72E+071.36E−04 2.88E−12 VLres08HVmut08 4.82E+07 1.44E−04 2.99E−12VLres08VHres16 4.61E+07 1.50E−04 3.25E−12 VLres10HVmut02 3.58E+071.22E−04 3.41E−12 VLres10HVmut04 4.01E+07 1.28E−04 3.20E−12VLres10HVmut08 4.73E+07 1.45E−04 3.06E−12 VLres10HVmut10 5.87E+071.29E−04 2.19E−12 VLres10VHres16 4.23E+07 1.51E−04 3.57E−12VLres14HVmut07 4.18E+07 1.25E−04 2.99E−12 VLres14HVmut08 4.37E+071.18E−04 2.71E−12 VLres14HVmut10 4.35E+07 1.24E−04 2.84E−12VLres14VHres16 4.03E+07 1.33E−04 3.30E−12 VLres16HVmut07 3.93E+071.25E−04 3.19E−12 VLres16HVmut10 4.17E+07 1.20E−04 2.88E−12VLres16VHres16 3.77E+07 1.39E−04 3.69E−12 Ch18C1 3.88E+07 1.02E−042.62E−12

From the above results, it was revealed that the produced anti-BMP10humanized antibodies have a binding activity equivalent to that of thech18C1 antibody.

[Example 14] Comparison of Neutralizing Activity Between Anti-BMP10Humanized Antibody and ch18C1 Antibody 14-1) Evaluation of NeutralizingActivity of Anti-BMP10 Humanized Antibody Against BMP10 Homodimer

With respect to the obtained anti-BMP10 humanized antibodies and thech18C1 antibody, the neutralizing activities against BMP10 were comparedusing the human ALK1 expressing reporter cells produced in Example 3,3-1).

To a 96-well fluorescence and luminescence plate (manufactured byCorning, Inc., Cat #3916), a human BMP10 mature dimer (manufactured by R& D Systems, Inc., Cat #2926-BP) was added at a final concentration of0.3 ng/mL, and subsequently, the anti-BMP10 humanized antibody obtainedin Example 12 or the ch18C1 antibody was prepared at 7 concentrations by3-fold serial dilution from the final concentration of 3000 ng/mL, andthen added.

Thereafter, an ALK1/Id1-Luc/CHO cell liquid in which the cells weresuspended in Excell 325 medium [Excell 325 PF CHO (manufactured by SAFC,Inc., Cat #14340C-1000 mL), 4 mM L-glutamine, 1×Penicillin,1×Streptomycin (manufactured by Nacalai, Inc., Cat #09367-34), 0.5 mg/mLhygromycin] was added at 5×10⁴ cells/well. After all samples were added,the liquid in the well was made uniform with a plate mixer, and thecells were cultured at 37° C. for 20 hours.

After 20 hours, an assay solution of Nano-Glo Luciferase Assay(manufactured by Promega Corporation, Cat #N1120) prepared according tothe package insert was added at 40 μL/well, followed by stirring, andthen, a luciferase activity was measured using Glomax (manufactured byPromega Corporation). The neutralizing activity (%) of the antibody wascalculated by setting the value of the well in which only the BMP10mature dimer was added without adding an antibody to be 0%, and thevalue of the well in which only Excell 325 medium was added withoutadding an antibody to be 100%. The results are shown in FIGS. 20 to 25.

As shown in FIGS. 20 to 25, all the produced anti-BMP10 humanizedantibodies exhibited a neutralizing activity equivalent to that of thech18C1 antibody.

14-2) Evaluation of Neutralizing Activity of Anti-BMP10 HumanizedAntibody Against BMP9/BMP10 Heterodimer in Human Blood

With respect to the obtained anti-BMP10 humanized antibodies and thech18C1 antibody, the neutralizing activities against a BMP9/BMP10heterodimer in human blood were compared using human ALK1 expressingreporter cells. As the human ALK1 expressing reporter cells, the cellsproduced in Example 3, 3-1) were used.

To a 96-well fluorescence and luminescence plate (manufactured byCorning, Inc., Cat #3916), human serum (Human True A serum, pool ofdonors, manufactured by Biopredic International, Cat #SER019) was addedat a final concentration of 10%.

Subsequently, the anti-BMP10 humanized antibody or the ch18C1 antibodywas added at a final concentration of 1000 ng/mL. Thereafter, anALK1/Id1-Luc/CHO cell suspension was added at 5×10⁴ cells/well. In allwells, Excell 325 medium was added so that the liquid amount became 100μL/well. After the liquid in the well was made uniform with a platemixer, the cells were cultured at 37° C. for 20 hours.

After 20 hours, an assay solution of Nano-Glo Luciferase Assay preparedaccording to the package insert was added at 40 μL/well, followed bystirring, and then, a luciferase activity was measured using Glomax(manufactured by Promega Corporation).

As shown in FIG. 26, all the produced anti-BMP10 humanized antibodiesexhibited a neutralizing activity equivalent to that of the ch18C1antibody against the BMP9/BMP10 heterodimer in human blood.

[Example 15] Evaluation of BMP9/BMP10 Heterodimer Neutralizing Activityon Blood Pressure of Normal Animal

An effect on blood pressure when neutralizing a BMP9/BMP10 heterodimerwas evaluated using SD rats. Male SD rats (manufactured by Charles RiverLaboratories Japan, Inc.) at 6 weeks of age were purchased and used foran experiment. The rats were given sterile tap water as drinking waterand chow FR-2 (manufactured by Funabashi Farm Co., Ltd.) as feed adlibitum. After acclimation, the rats underwent surgery for implantationof a telemetry transmitter at 7 weeks of age. The method for the surgeryfor implantation of a telemetry transmitter and the acquisition of theblood pressure waveform were carried out according to Example 7.

In a BMP9 antibody administration group, a solution in which the BMP9antibody 10D5 was prepared at 10 mg/mL with PBS was administered at adose of 1 mL/kg. In a BMP9/BMP10 heterodimer neutralization group, asolution in which the BMP9 antibody 10D5 and the BMP10 antibody 11H10were mixed and prepared at a final concentration of 10 mg/mL and 5mg/mL, respectively, with PBS was administered at a dose of 1 mL/kg. Theadministration was carried out through a subcutaneous route.

As shown in FIG. 27A, as compared with the case when the vehicle wasadministered, a change in the systolic blood pressure by theadministration of the 10D5 antibody was not observed. On the other hand,as shown in FIG. 27B, as compared with the case when the vehicle wasadministered, in the BMP9/BMP10 heterodimer neutralization group inwhich the 10D5 antibody and the 11H10 antibody were mixed andadministered, a decrease in the systolic blood pressure was observed.

A change in the systolic blood pressure is not caused by either of the10D5 antibody and the 11H10 antibody alone. However, it was newly foundthat when the 10D5 antibody and the 11H10 antibody are used incombination, by the neutralization of the BMP9/BMP10 heterodimer, anantihypertensive action is exhibited.

[Example 16] Evaluation of Effect of Human BMP10 Homodimer on BloodPressure of Normal Animal 16-1) Preparation of Human BMP10 RecombinantProtein

In order to evaluate the effect of a human BMP10 homodimer on bloodpressure, a human BMP10 recombinant protein was prepared. A human BMP10expression vector was prepared according to the method described inExample 20 in WO 2014/007198. Further, a human Furin expression plasmidwas prepared by incorporating a human Furin full-length cDNA into apEAK8 vector (Edge Biosystems, Inc.) using In-Fusion HD Cloning Kit(manufactured by Takara Bio, Inc.).

Transient expression was carried out for human BMP10 and human Furinusing EXPI 293 Expression system (manufactured by Thermo FisherScientific, Inc.), whereby a human BMP10 recombinant protein wasexpressed. The culture supernatant was obtained from the culturesolution by centrifugation and filtration using a 0.22 μm filter.

Subsequently, each protein was purified using Ni-NTA Agarose(manufactured by QIAGEN, Inc.). As a binding buffer, 20 mM HEPES-NaOH(pH 7.4) containing 500 mM NaCl and 40 mM imidazole was used, and as anelution buffer, 20 mM HEPES-NaOH (pH 7.4) containing 500 mM NaCl and 200mM imidazole was used.

The buffer was replaced with PBS using a NAP-25 column (manufactured byGE Healthcare, Inc., 17-0852-02). An absorbance at 280 nm was measuredto determine the concentration of each protein solution. As a molecularextinction coefficient, 0.96 mL/(mg·cm) was used. The thus obtainedBMP10 recombinant protein does not contain a full-length protein, butcontains only a mature protein and an N-terminal propeptide protein,however, it forms a homodimer in the same manner as the BMP10recombinant protein containing a full-length protein produced by themethod of Example 1-1 and has a function as BMP10.

16-2) Evaluation of Effect of BMP10 Recombinant Protein on BloodPressure

An effect on blood pressure when administering the human BMP10recombinant protein was evaluated using SD rats. Male SD rats(manufactured by Charles River Laboratories Japan, Inc.) at 6 weeks ofage were purchased and used for an experiment. The rats were givensterile tap water as drinking water and chow FR-2 (manufactured byFunabashi Farm Co., Ltd.) as feed ad libitum. After acclimation, therats underwent surgery for implantation of a telemetry transmitter at 7weeks of age.

The method for the surgery for implantation of a telemetry transmitterand the acquisition of the blood pressure waveform were carried outaccording to Example 7. A solution in which the human BMP10 recombinantprotein obtained in Example 16-1 was prepared at 0.5 mg/mL with PBS wasadministered at a dose of 1 mL/kg. The administration was carried outthrough an intravenous route.

As shown in FIG. 28, as compared with the case when the vehicle wasadministered, an increase in the systolic blood pressure by theadministration of the human BMP10 recombinant protein was observed.

From the above results, it was newly found that the human BMP10homodimer has a vasopressor effect.

While the present invention has been described in detail with referenceto specific embodiments, it will be apparent to those skilled in the artthat various changes and modifications can be made without departingfrom the spirit and scope of the present invention. Note that thepresent application is based on Japanese Patent Application (JapanesePatent Application No. 2017-238106) filed on Dec. 12, 2017, which isincorporated by reference in its entirety.

Sequence Listing Free Text

SEQ ID NO: 1: nucleotide sequence of primer RV1 specific to rat IgG1

SEQ ID NO: 2: nucleotide sequence of primer RV1 specific to rat IgG2a

SEQ ID NO: 3: nucleotide sequence of primer RV1 specific to rat Ig(κ)

SEQ ID NO: 4: nucleotide sequence of primer RV1 specific to rat Ig(λ)

SEQ ID NO: 5: entire nucleotide sequence of VH of 18C1 antibody

SEQ ID NO: 6: entire nucleotide sequence of VH of 12H3 antibody

SEQ ID NO: 7: entire nucleotide sequence of VH of 11H10 antibody

SEQ ID NO: 8: entire amino acid sequence of VH of 18C1 antibody(including signal sequence)

SEQ ID NO: 9: entire amino acid sequence of VH of 12H3 antibody(including signal sequence)

SEQ ID NO: 10: entire amino acid sequence of VH of 11H10 antibody(including signal sequence)

SEQ ID NO: 11: entire nucleotide sequence of VL of 18C1 antibody

SEQ ID NO: 12: entire nucleotide sequence of VL of 12H3 antibody

SEQ ID NO: 13: entire nucleotide sequence of VL of 11H10 antibody

SEQ ID NO: 14: entire amino acid sequence of VL of 18C1 antibody(including signal sequence)

SEQ ID NO: 15: entire amino acid sequence of VL of 12H3 antibody(including signal sequence)

SEQ ID NO: 16: entire amino acid sequence of VL of 11H10 antibody(including signal sequence)

SEQ ID NO: 17: nucleotide sequence of VH of 18C1 antibody (excludingsignal sequence)

SEQ ID NO: 18: nucleotide sequence of VH of 12H3 antibody (excludingsignal sequence)

SEQ ID NO: 19: nucleotide sequence of VH of 11H10 antibody (excludingsignal sequence)

SEQ ID NO: 20: nucleotide sequence of VL of 18C1 antibody (excludingsignal sequence)

SEQ ID NO: 21: nucleotide sequence of VL of 12H3 antibody (excludingsignal sequence)

SEQ ID NO: 22: nucleotide sequence of VL of 11H10 antibody (excludingsignal sequence)

SEQ ID NO: 23: amino acid sequence of VH of 18C1 antibody (excludingsignal sequence)

SEQ ID NO: 24: amino acid sequence of VH of 12H3 antibody (excludingsignal sequence)

SEQ ID NO: 25: amino acid sequence of VH of 11H10 antibody (excludingsignal sequence)

SEQ ID NO: 26: amino acid sequence of VL of 18C1 antibody (excludingsignal sequence)

SEQ ID NO: 27: amino acid sequence of VL of 12H3 antibody (excludingsignal sequence)

SEQ ID NO: 28: amino acid sequence of VL of 11H10 antibody (excludingsignal sequence)

SEQ ID NO: 29: amino acid sequence of CDR1 of VH of 18C1 antibody

SEQ ID NO: 30: amino acid sequence of CDR2 of VH of 18C1 antibody

SEQ ID NO: 31: amino acid sequence of CDR3 of VH of 18C1 antibody

SEQ ID NO: 32: amino acid sequence of CDR1 of VL of 18C1 antibody

SEQ ID NO: 33: amino acid sequence of CDR2 of VL of 18C1 antibody

SEQ ID NO: 34: amino acid sequence of CDR3 of VL of 18C1 antibody

SEQ ID NO: 35: amino acid sequence of CDR1 of VH of 12H3 antibody

SEQ ID NO: 36: amino acid sequence of CDR2 of VH of 12H3 antibody

SEQ ID NO: 37: amino acid sequence of CDR3 of VH of 12H3 antibody

SEQ ID NO: 38: amino acid sequence of CDR1 of VL of 12H3 antibody

SEQ ID NO: 39: amino acid sequence of CDR2 of VL of 12H3 antibody

SEQ ID NO: 40: amino acid sequence of CDR3 of VL of 12H3 antibody

SEQ ID NO: 41: amino acid sequence of CDR1 of VH of 11H10 antibody

SEQ ID NO: 42: amino acid sequence of CDR2 of VH of 11H10 antibody

SEQ ID NO: 43: amino acid sequence of CDR3 of VH of 11H10 antibody

SEQ ID NO: 44: amino acid sequence of CDR1 of VL of 11H10 antibody

SEQ ID NO: 45: amino acid sequence of CDR2 of VL of 11H10 antibody

SEQ ID NO: 46: amino acid sequence of CDR3 of VL of 11H10 antibody

SEQ ID NO: 47: amino acid sequence of human BMP10 protein (includingsignal sequence)

SEQ ID NO: 48: amino acid sequence of human BMP10 mature region

SEQ ID NO: 49: nucleotide sequence encoding human BMP10 protein(including signal sequence)

SEQ ID NO: 50: nucleotide sequence encoding human BMP10 mature region

SEQ ID NO: 51: amino acid sequence of human BMPRII

SEQ ID NO: 52: amino acid sequence of human ALK1

SEQ ID NO: 53: nucleotide sequence of primer Fwd used for amplificationof light chain of 18C1 antibody

SEQ ID NO: 54: nucleotide sequence of primer Rv used for amplificationof light chain of 18C1 antibody

SEQ ID NO: 55: nucleotide sequence of primer Fwd used for amplificationof heavy chain of 18C1 antibody

SEQ ID NO: 56: nucleotide sequence of primer Rv used for amplificationof heavy chain of 18C1 antibody

SEQ ID NO: 57: nucleotide sequence of primer Fwd used for amplificationof light chain of 12H3 antibody

SEQ ID NO: 58: nucleotide sequence of primer Rv used for amplificationof light chain of 12H3 antibody

SEQ ID NO: 59: nucleotide sequence of primer Fwd used for amplificationof heavy chain of 12H3 antibody

SEQ ID NO: 60: nucleotide sequence of primer Rv used for amplificationof heavy chain of 12H3 antibody

SEQ ID NO: 61: nucleotide sequence of primer Fwd used for amplificationof light chain of 11H10 antibody

SEQ ID NO: 62: nucleotide sequence of primer Rv used for amplificationof light chain of 11H10 antibody

SEQ ID NO: 63: nucleotide sequence of primer Fwd used for amplificationof heavy chain of 11H10 antibody

SEQ ID NO: 64: nucleotide sequence of primer Rv used for amplificationof heavy chain of 11H10 antibody

SEQ ID NO: 65: amino acid sequence of human BMP9 mature region

SEQ ID NO: 66: amino acid sequence of human BMP9 protein (includingsignal sequence)

SEQ ID NO: 67: nucleotide sequence encoding human BMP9 protein(including signal sequence)

SEQ ID NO: 68: nucleotide sequence encoding human BMP9 mature region

SEQ ID NO: 69: amino acid sequence of human Endoglin

SEQ ID NO: 70: amino acid sequence of HV0 of humanized 18C1 antibody

SEQ ID NO: 71: amino acid sequence of LV0 of humanized 18C1 antibody

SEQ ID NO: 72: amino acid sequence of VLres01 of humanized 18C1 antibody

SEQ ID NO: 73: amino acid sequence of VLres02 of humanized 18C1 antibody

SEQ ID NO: 74: amino acid sequence of VLres03 of humanized 18C1 antibody

SEQ ID NO: 75: amino acid sequence of VLres04 of humanized 18C1 antibody

SEQ ID NO: 76: amino acid sequence of VLres05 of humanized 18C1 antibody

SEQ ID NO: 77: amino acid sequence of VLres06 of humanized 18C1 antibody

SEQ ID NO: 78: amino acid sequence of VLres07 of humanized 18C1 antibody

SEQ ID NO: 79: amino acid sequence of VLres08 of humanized 18C1 antibody

SEQ ID NO: 80: amino acid sequence of VLres09 of humanized 18C1 antibody

SEQ ID NO: 81: amino acid sequence of VLres10 of humanized 18C1 antibody

SEQ ID NO: 82: amino acid sequence of VLres11 of humanized 18C1 antibody

SEQ ID NO: 83: amino acid sequence of VLres12 of humanized 18C1 antibody

SEQ ID NO: 84: amino acid sequence of VLres13 of humanized 18C1 antibody

SEQ ID NO: 85: amino acid sequence of VLres14 of humanized 18C1 antibody

SEQ ID NO: 86: amino acid sequence of VLres15 of humanized 18C1 antibody

SEQ ID NO: 87: amino acid sequence of VLres16 of humanized 18C1 antibody

SEQ ID NO: 88: amino acid sequence of HVmut01 of humanized 18C1 antibody

SEQ ID NO: 89: amino acid sequence of HVmut02 of humanized 18C1 antibody

SEQ ID NO: 90: amino acid sequence of HVmut03 of humanized 18C1 antibody

SEQ ID NO: 91: amino acid sequence of HVmut04 of humanized 18C1 antibody

SEQ ID NO: 92: amino acid sequence of HVmut05 of humanized 18C1 antibody

SEQ ID NO: 93: amino acid sequence of HVmut06 of humanized 18C1 antibody

SEQ ID NO: 94: amino acid sequence of HVmut07 of humanized 18C1 antibody

SEQ ID NO: 95: amino acid sequence of HVmut08 of humanized 18C1 antibody

SEQ ID NO: 96: amino acid sequence of HVmut09 of humanized 18C1 antibody

SEQ ID NO: 97: amino acid sequence of HVmut10 of humanized 18C1 antibody

SEQ ID NO: 98: amino acid sequence of VHres16 of humanized 18C1 antibody

SEQ ID NO: 99: amino acid sequence of CDR2 of HVmut05 to 10 of humanized18C1 antibody

1. A monoclonal antibody, comprising: a heavy chain comprisingcomplementarity determining regions (hereinafter abbreviated as CDRs) 1and 3 comprising amino acid sequences represented by SEQ ID NOs: 29 and31, respectively, and CDR2 comprising an amino acid sequence representedby SEQ ID NO: 30 or an amino acid sequence in which serine at position16 of the amino acid sequence represented by SEQ ID NO: 30 issubstituted with aspartic acid; and a light chain comprising CDRs 1 to 3comprising amino acid sequences represented by SEQ ID NOs: 32 to 34,respectively, or an antibody fragment thereof.
 2. The monoclonalantibody or the antibody fragment thereof according to claim 1,comprising: a light chain variable region (hereinafter abbreviated asVL) comprising any one amino acid sequence selected from SEQ ID NOs: 71to 87 and/or a heavy chain variable region (hereinafter abbreviated asVH) comprising any one amino acid sequence selected from SEQ ID NOs: 70and 88 to
 98. 3. The monoclonal antibody or the antibody fragmentthereof according to claim 1 selected from the following (a) to (w): (a)a monoclonal antibody comprising VH comprising an amino acid sequencerepresented by SEQ ID NO: 94 and VL comprising an amino acid sequencerepresented by SEQ ID NO: 73, or an antibody fragment thereof; (b) amonoclonal antibody including VH comprising an amino acid sequencerepresented by SEQ ID NO: 95 and VL comprising an amino acid sequencerepresented by SEQ ID NO: 73, or an antibody fragment thereof; (c) amonoclonal antibody including VH comprising an amino acid sequencerepresented by SEQ ID NO: 91 and VL comprising an amino acid sequencerepresented by SEQ ID NO: 75, or an antibody fragment thereof; (d) amonoclonal antibody comprising VH comprising an amino acid sequencerepresented by SEQ ID NO: 98 and VL comprising an amino acid sequencerepresented by SEQ ID NO: 75, or an antibody fragment thereof; (e) amonoclonal antibody comprising VH comprising an amino acid sequencerepresented by SEQ ID NO: 89 and VL comprising an amino acid sequencerepresented by SEQ ID NO: 77, or an antibody fragment thereof; (f) amonoclonal antibody comprising VH comprising an amino acid sequencerepresented by SEQ ID NO: 97 and VL comprising an amino acid sequencerepresented by SEQ ID NO: 77, or an antibody fragment thereof; (g) amonoclonal antibody comprising VH comprising an amino acid sequencerepresented by SEQ ID NO: 97 and VL comprising an amino acid sequencerepresented by SEQ ID NO: 78, or an antibody fragment thereof; (h) amonoclonal antibody comprising VH comprising an amino acid sequencerepresented by SEQ ID NO: 98 and VL comprising an amino acid sequencerepresented by SEQ ID NO: 78, or an antibody fragment thereof; (i) amonoclonal antibody comprising VH comprising an amino acid sequencerepresented by SEQ ID NO: 91 and VL comprising an amino acid sequencerepresented by SEQ ID NO: 79, or an antibody fragment thereof; (j) amonoclonal antibody comprising VH comprising an amino acid sequencerepresented by SEQ ID NO: 95 and VL comprising an amino acid sequencerepresented by SEQ ID NO: 79, or an antibody fragment thereof; (k) amonoclonal antibody comprising VH comprising an amino acid sequencerepresented by SEQ ID NO: 98 and VL comprising an amino acid sequencerepresented by SEQ ID NO: 79, or an antibody fragment thereof; (l) amonoclonal antibody comprising VH comprising an amino acid sequencerepresented by SEQ ID NO: 89 and VL comprising an amino acid sequencerepresented by SEQ ID NO: 81, or an antibody fragment thereof; (m) amonoclonal antibody comprising VH comprising an amino acid sequencerepresented by SEQ ID NO: 91 and VL comprising an amino acid sequencerepresented by SEQ ID NO: 81, or an antibody fragment thereof; (n) amonoclonal antibody comprising VH comprising an amino acid sequencerepresented by SEQ ID NO: 95 and VL comprising an amino acid sequencerepresented by SEQ ID NO: 81, or an antibody fragment thereof; (o) amonoclonal antibody comprising VH comprising an amino acid sequencerepresented by SEQ ID NO: 97 and VL comprising an amino acid sequencerepresented by SEQ ID NO: 81, or an antibody fragment thereof; (p) amonoclonal antibody comprising VH comprising an amino acid sequencerepresented by SEQ ID NO: 98 and VL comprising an amino acid sequencerepresented by SEQ ID NO: 81, or an antibody fragment thereof; (q) amonoclonal antibody comprising VH comprising an amino acid sequencerepresented by SEQ ID NO: 94 and VL comprising an amino acid sequencerepresented by SEQ ID NO: 85, or an antibody fragment thereof; (r) amonoclonal antibody comprising VH comprising an amino acid sequencerepresented by SEQ ID NO: 95 and VL comprising an amino acid sequencerepresented by SEQ ID NO: 85, or an antibody fragment thereof; (s) amonoclonal antibody comprising VH comprising an amino acid sequencerepresented by SEQ ID NO: 97 and VL comprising an amino acid sequencerepresented by SEQ ID NO: 85, or an antibody fragment thereof; (t) amonoclonal antibody comprising VH comprising an amino acid sequencerepresented by SEQ ID NO: 98 and VL comprising an amino acid sequencerepresented by SEQ ID NO: 85, or an antibody fragment thereof; (u) amonoclonal antibody comprising VH comprising an amino acid sequencerepresented by SEQ ID NO: 94 and VL comprising an amino acid sequencerepresented by SEQ ID NO: 87, or an antibody fragment thereof; (v) amonoclonal antibody comprising VH comprising an amino acid sequencerepresented by SEQ ID NO: 97 and VL comprising an amino acid sequencerepresented by SEQ ID NO: 87, or an antibody fragment thereof; and (w) amonoclonal antibody comprising VH comprising an amino acid sequencerepresented by SEQ ID NO: 98 and VL comprising an amino acid sequencerepresented by SEQ ID NO: 87, or an antibody fragment thereof.
 4. Themonoclonal antibody or the antibody fragment thereof according to claim1, having a neutralizing activity against BMP10.
 5. The monoclonalantibody or the antibody fragment thereof according to claim 1, having aneutralizing activity against a BMP9/BMP10 heterodimer.
 6. Themonoclonal antibody or the antibody fragment thereof according to claim1, which is a genetically recombinant antibody.
 7. The antibody fragmentaccording to claim 1, which is an antibody fragment selected from a Fab,a Fab′, a (Fab′)₂, a single chain antibody (scFv), a dimerized V region(diabody), a disulfide stabilized V region (dsFv), and a peptidecomprising a CDR.
 8. A DNA, encoding the monoclonal antibody or theantibody fragment thereof according to claim
 1. 9. A recombinant vector,comprising the DNA according to claim
 8. 10. A transformant, obtained byintroducing the recombinant vector according to claim 9 into a hostcell.
 11. A method for producing a monoclonal antibody or an antibodyfragment thereof, comprising: culturing the transformant according toclaim 10 in a culture medium, and collecting the antibody or theantibody fragment from the culture.
 12. A therapeutic method forhypertension and/or a hypertensive disease, comprising administering atherapeutically-effective amount an antagonist for at least one of BMP10and a BMP9/BMP10 heterodimer to a subject in need thereof.
 13. Thetherapeutic method according to claim 12, wherein the antagonist for atleast one of BMP10 and a BMP9/BMP10 heterodimer is administeredconcurrently or sequentially with a BMP9 antagonist. 14-18. (canceled)19. A method selected from one of following (a) or (b) comprising a useof an antagonist for at least one of BMP10 and a BMP9/BMP10 heterodimer:(a) a diagnostic method for a disease associated with human BMP10 (b) animmunological detection method or a measurement method for human BMP10.